DE60219592T2 - INTRINSIC FLUORESCENT, SELF-MULTIMERIZING MHC FUSION PROTEINS, AND ITS COMPLEXES - Google Patents

Abstract

Presented are intrinsically fluorescent, self-multimerizing MHC fusion proteins, and complexes assembled therefrom that are capable of detectably labeling antigen-specific T lymphocytes. Also presented are methods for labeling antigen-specific T lymphocytes with the intrinsically fluorescent complexes of the present invention, and methods, particularly flow cytometric methods, for detecting, enumerating, enriching, and depleting antigen specific T lymphocytes so labeled.

Description

BACKGROUND OF THE INVENTION

Soluble tetramers of major histocompatibility complex (MHC) proteins, which is loaded with specific peptide antigen and fluorescently labeled are ("MHC tetramers"), have become in the last five Years as extraordinary useful in the detection, the counting, Characterization and purification of antigen-specific T lymphocytes. Applications of MHC class I-tetramers were recently surveyed in Doherty et al., Annu. Rev. Immunol. 18: 561-92 (2000); Ogg et al., Immunol. Lett. 66 (1-3): 77-80 (1999); Maini et al., Immunol. Today 20 (6): 262-6 (1999); and Doherty, Curr. Opin. Microbiol. 1 (4): 419-22 (1998). applications MHC class 11 tetramers are described in Reichstetter et al., J. Immunol. 165 (12): 6994-8 (2000); Kwok et al., J. Immunol. 164 (8): 4244-9 (2000); Liu et al., Proc. Natl. Acad. Sci. USA, 97 (26): 14596-14601 (2000); Novak et al., J. Clin. Invest. 104 (12): R63-7 (1999); Crawford et al., Immunity 8: 675-682 (1998), and Kozono et al., Nature 369: 151-154 (1994), discussed.

MHC tetramers bind to the T cell receptor ("TCR") of T lymphocytes in an antigen and MHC-specific manner; see, for. Altman et al., Science 274: 94 (1996), and U.S. Patent No. 5,635,363.

The specificity of the staining reagent is mediated by both the MHC structural unit and the peptide included within the tetrameric complex. Thus, tetramers comprising the extracellular domains of MHC class I α-chain molecules complexed to β2-microglobulin and loaded with antigenic peptides of about 9 amino acids are restricted to class I, typically CD8 ⁺ . , T cells that are specific for the loading peptide and the MHC allele. Tetramers comprising four class II heterodimers - each comprising the extracellular domains of MHC class II α and class II β chain extracellular domains - loaded with antigenic peptides are attached to class II peptides. II-restricted, typically CD4 ⁺ , T cells bind in an antigen-specific and MHC-limiting manner.

A avidity or binding ability, which is sufficient to form a stable bond to allow the T lymphocytes, results from the multimerization of the MHC / peptide complex. To maintain self-tolerance is the natural one affinity from TCR for MHC / peptide low, and too low to use one monovalent MHC / peptide complex as an immunological stain allow; Matsui et al., Science 254: 1788-91 (1991); Matsui et al., Proc. Natl. Acad. Sci. USA, 91: 12862-6 (1994). The multimerization elevated the binding ability of the MHC / peptide complex is sufficient for TCR, to allow a stable bond. Typically, the multimerization by enzymatic biotinylation of a BirA substrate peptide, which artificially achieved in the MHC chain was achieved; the biotinylated MHC chain binds afterwards Avidin or streptavidin with a 4: 1 molar stoichiometry.

The MHC tetramer is activated by direct or indirect conjugation Fluorophore made detectable. Typically, a direct Conjugation is accomplished by multimerizing the MHC / peptide molecules under Use of a streptavidin or avidin molecule previously attached to a fluorophore has been conjugated. Such avidin and streptavidin proteins are commercially available from a variety of suppliers (e.g. B. Becton Dickinson Immunocytometry Systems, San Jose, CA, USA; Biomeda, Foster City, CA, USA; Ancell Corp., Bayport, MN, USA; Southern Biotechnology Assocs., Inc., Birmingham, AL, USA). An indirect one Labeling can be performed using a fluorophore-conjugated antibody, of specificity for the Avidin / streptavidin residue or for has non-polymorphic determinants of the multimerized MHC chain.

Even though MHC tetramers proved to be extremely useful certain aspects of their synthesis are a problem.

A correct multimerization requires the controlled stoichiometric, preliminary biotinylation of the MHC chain and further requires the subsequent one stoichiometric Binding of biotinylated chains to avidin or streptavidin. Inefficiency or Inaccuracy in one or both influenced by these steps the yield and applicability of the resulting tetrameric complex.

To the An example is an inefficient biotinylation of the MHC chain - d. H. the failure to incorporate at least one biotin per MHC molecule - one reduced yield of tetramers. The inefficient removal of non-conjugated biotin from the reaction products, with carry from non-conjugated biotin molecules into the multimerization reaction, can saturate avidin molecules and also reduce the tetramer yield.

Biotinylation of MHC molecules at multiple sites can reduce the binding affinity of the resulting tetrameric complexes. Previous attempts at multimerization using chemical biotinylation failed, possibly because of the multiple and random chemical modification of MHC molecules poor and often inactive orientation of the MHC / peptide moieties in the multimerized complex. Although enzymatic biotinylation has improved the control of the biotinylation process, enzymatic modification itself is subject to the well-known alternations, including dependence on enzyme concentration, substrate concentration, substrate accessibility, enzyme activity, and the like; multiple enzymatic biotinylation can mimic the low binding affinity observed with the earlier chemically biotinylated complexes.

The Biotinylation at multiple sites of the MHC chain, combined with the inevitable Use of an extrinsic multimerization moiety, such as avidin, also does the disturbing Generation of multimers higher Order while the multimerization reaction possible. Even if a small number of MHC / peptide subunits are two or more contains several biotins a cross-linking of the avidin / streptavidin structural unit take place. Such multimers are higher Order a decreased attachment pleasure due to steric hindrance show and can to be swept, under other circumstances, to complexes higher Lead order, which one increased Have willingness to bond. Variations in the willingness to bond, whether reductions or increases from the mean, you can too Variations in staining intensity cause which not with TCR density or antigen affinity being related. Multimers of higher order can also generate a more intense fluorescence signal, which is also confusing Fluctuations in the color intensity leads.

Vice versa can the carry of non-conjugated biotin molecules in the multimerization reaction to a partial saturation of avidin / streptavidin structural units with unconjugated biotin, resulting in the formation of multimeric complexes containing less than four MHC / peptide subunits exhibit. Such lower order complexes may be one lower binding affinity for TCR show what leads to variations in the color intensity, which not related to TCR density or antigen / MHC affinity.

The Requirement for a separate fluorophore conjugation step also offers opportunities for Loss of yield and unintended variation either in terms of the avidity for TCR or the fluorescence intensity, or both.

In a related approach will be MHC / Ig chimeras instead Tetramers from MHC, used to produce antigen-specific T lymphocytes to mark. Dal Porto et al., Proc. Natl. Acad. Sci. USA 90: 6671-6675 (1993); Greten et al., Proc. Natl. Acad. Sci. USA 95: 7568-7573 (1998); Hamad et al., J. Exp. Med. 188: 1633-1640 (1998); Schneck et al., U.S. Patent Nos. 6,015,884 and 6,140,113.

In Class I chimeras be the extracellular domains the MHC class Iα chain in reading frame fused into the variable region of an IgG heavy chain. Under suitable perform oxidizing conditions the heavy chain chimeras are a self-dimerization by disulfide bonds: dimerization mediates avidity for TCR, which is sufficient to the application of the dimeric fusion protein as a T-cell labeling reagent to allow. As with MHC tetramers, the dimeric class I chimera molecule must be associated with β2 microglobulin and loaded with specific antigenic peptide for recognition to allow for antigen-specific T cells; unlike MHC tetramers, have to the MHC / Ig chimera molecules in addition the application associated with Ig light chains.

In Class II MHC / Ig chimeras are the extracellular domains of MHC class II α and β chains separately Ig heavy and light chain subunits typically, the β-subunit domains are linked to IgG heavy chain and the class II α domains on Ig light chain, typically κ, are merged. As with MHC tetramers, the class II chimera molecule must be specific loaded antigenic peptide to the detection of antigen-specific To allow T cells; For the stability may antigenic peptide directly to the N-terminus of the class II β-subunit be merged; Kozono et al., Nature 369: 151-154 (1994); Liu et al., Proc. Natl. Acad. Sci. USA 97: 14596-14601 (2000).

The Marking the dimeric chimeras is typically using a fluorophore-conjugated Anti-Ig secondary antibody accomplished.

The self-dimerizing chimeras do the biotinylation step and use an extrinsic Multimerization unit derived from MHC chimeras required, with their accompanying problems unnecessary. The allow dimeric chimeras also a simpler post-synthesis loading of antigenic peptide.

Indeed are the dimeric chimeras not without their own problems.

To the First, the dimer valence may have a lower binding affinity for TCR cause when they are obtained by using tetramers would, what the ability reduces or eliminates low affinity TCR labeling.

To the Second does the extra Requirement of Ig light chain association - whether native Ig light chains, as in the class I chimeras, or recombinant Ig light chain fusion proteins, as in class II chimeras - coexpression several recombinant expression constructs in a single Host cell, or, in the case of class I chimeras, the use of a host cell line, which constitutively expressed Ig light chain, necessary.

To the Third, although self-dimerization is the biotinylation step and resting on an extrinsic multimerization moiety of Picking up MHC tetramers, where the yield of multimers of known valence (complexity) is potentially can be improved the dimers dissociate under reducing conditions.

Furthermore brings any of the strategies to make the dimeric chimera fluorescently detectable to make trouble with yourself.

To the For example, the use of secondary antibodies to visualize a simultaneous Make detection of multiple antigens more difficult; this can be a significant Problem if, as is often the case, the population of antigen-specific T cells is small, which necessitates the use of multiple parameters, to detect and count the cells correctly. A direct chemical Conjugation of fluorophore to the chimera, an alternative to use from secondary antibodies can affinity interactions the chimera with their associated Disturb or TCR even pick up. Direct chemical conjugation may also be used chimeric molecules to lead, which different molar ratios have marker, which is flow cytometric or other fluorescence-based Makes analysis more problematic.

Therefore There is a need for reagents that can be used to To mark T lymphocytes based on their antigen (and MHC) specificity, which have sufficient binding ability to produce a stable Binding to T lymphocytes through interactions with surface TCR but which are not biotinylated and extrinsic Require multimerization; there is a corresponding one Need for antigen-specific T-cell labeling reagents which, without Requirement of using secondary antibodies or direct chemical Conjugation to a fluorophore, can be made fluorescent.

The substrate-independent, self-fluorescent green fluorescent protein from Aequorea victoria ("GFP") is in the flow cytometric ("FACS") analysis of cells have been used which GFP, or GFP fusions, within the cell expressing; this is summarized in a clear overview in Galbraith et al., Flow cytometric analysis and FACS sorting cells based on GFP accumulation ", Methods Cell Biol. 58: 315-41 (1999). GFP variants which have features that for the Flow cytometry are improved have been described; U.S. Patents Nos. 6,090,919 and 5,804,387; Cormack et al., Gene 173: 33-38 (1996). GFP and GFP variants are typically more for internal labeling of Cells as for the external labeling of cell surface structures has been used.

One removed homolog of GFP, designated DsRed (also called as drFP583) has recently been cloned from the coral Discosoma; Matz et al., Nature Biotechnol. 17: 969-973 (1999); Vectors which for the Excision cloning, bacterial expression and mammalian expression by DsRed, alone or as a fusion protein, are now commercially available (Clontech Laboratories, Inc., Palo Alto, CA, USA).

Natives DsRed has an excitation (absorption) maximum of about 558 nm and has an emission maximum of about 583 nm, an essential one Redshift from the GFP emission maximum of about 509 nm. DsRed Thus, the standard 488 nm laser line used in Flow cytometers is routinely available, stimulated and has an emission spectrum, which readily distinguishable from the autofluorescence background and from that of GFP is what makes DsRed a candidate for two-color flow cytometry applications with GFP or FACS optimized Makes mutants of GFP.

Full Investigations of the structure and spectral properties of DsRed, Baird et al., Proc. Natl. Acad. Sci. USA 97: 11984-11989 (2000); Gross et al., Proc. Natl. Acad. Sci. USA 97: 11990-11995 (2000); Heikal et al., Proc. Natl. Acad. Sci. USA 97: 11996-12001 (2000); Wall et al., Nature Structural Biol. 7: 1133-1138 (2000) but identifies two features that are claimed to be against the widespread use of DsRed in flow cytometry and other fluorescence analyzes.

To the First, the red fluorescence of DsRed matures only slowly, taking days be required to complete from green mature to red, both in vitro and in vivo. A maturation period in the order of magnitude By days, procedures will rule out DsRed as a reporter short-term gene expression or to use fusion proteins in organisms with short generation periods or to pursue a rapid development. Baird et al., Proc. Natl. Acad. Sci. USA 97: 11984-11989 (2000).

To the Second, DsRed seems an inevitable, and self-multimerizing, To be a tetramer; Baird et al., Proc. Natl. Acad. Sci. USA 97: 11984-11989 (2000); Wall et al., Nature Structural Biol. 7: 1133-1138 (2000); Gross et al., Proc. Natl. Acad. Sci. USA 97: 11990-11995 (2000). Although oligomerization is irrelevant to the application of DsRed as simple reporter for Gene expression can be serious problems in most lead to potential applications, where DsRed fuses to a host protein to "trafficking" or to track interactions of the latter. Continue to be many proteins in signal transduction by oligomerization activated; a fusion to DsRed could be a constitutive signal generation cause. For Host proteins, which are already oligomeric, could be the fusion to DsRed either Collisions in terms of stoichiometry, steric conflicts of quaternary Structures or networking to create massive aggregates.

in view of These "major disadvantages" will "any / many potential cell biological applications of DsRed the suppression of Require tetramerization and acceleration of maturation. "Baird et al., Proc. Natl. Acad. Sci. USA 97: 11984-11989 (2000). "For Most biotechnological applications are both the slow ones Maturation as well as the oligomerization of DsRed undesirable properties, which must be eliminated by systematic mutagenesis. ", Wall et al., Nature Structural Biol. 7: 1133-1138 (2000). "The excellent brightness and stability of DsRed and many potential applications for a long wavelength fluorescent protein provide a comprehensive justification for greater efforts to remedy these remaining shortcomings "Gross et al., Proc. Natl. Acad. Sci. USA 97: 11990-11995 (2000).

BRIEF DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide reagents which can be used to label T lymphocytes based on their antigen (and MHC) specificity, which have a sufficient binding ability for T-lymphocytes, to provide a stable bond to surface TCR without requirement of Biotinylation and application of an extrinsic multimerization moiety to allow and which can be made fluorescent, without Requirement of use of secondary antibodies or a direct chemical Conjugation to the fluorophore. It is another object of the invention Method of marking, detecting, counting, enriching, isolating, Depleting and activating antigen-specific T lymphocytes under To provide use of such reagents.

These and other requirements in the art are covered by the present Invention meets which a property - tetramerization - exploits, the asserted unfavorable in the newly discovered, self-fluorescent, self-multimerizing Fluorophore DsRed is, and which, by using the resulting Tetramers as an extracellular instead of an intracellular marker, from the other property claimed in the art to be detrimental to DsRed, namely the slow maturation of its protein fluorophore after translation.

In In a first aspect, the invention thus provides a recombinant A fusion protein ready comprising: a GFP-like chromophore; at least a multimerization domain; and an MHC peptide-presenting Structural unit or MHC peptide-presenting structural unit. Functionally, the fusion protein comprises means for fluorescing, wherein the fluorescers are completely within the amino acid sequence the protein are encoded; Means for multimerizing; and means, which to contribute to the presentation of a peptide antigen to an MHC-restricted T-lymphocyte.

In a preferred embodiment includes the fusion protein of the present invention from the N-terminus to the C-terminus, contiguous α1, α2 and α3 domains from a chosen one MHC class I α subunit, in which, in the reading frame, essentially the complete DsRed connects, which one both the GFP-like Fluorophore and provides two distinct multimerization domains. This preferred fusion protein tetramerizes spontaneously in solution.

If they are correctly multimerized and loaded with the peptide, the Fusion proteins of the present invention can be used without further direct or indirect conjugation to an exogenous fluorophore T lymphocytes based to fluorescently label for antigen (and MHC) specificity; The reagents of the present invention can therefore be used in all methods be used for which MHC tetramers and MHC / Ig chimera fusion proteins currently used.

The fusion proteins of the present invention are typically produced by recombinant expression in host cells. Thus, in further aspects, the present invention includes nucleic acids encoding the above-described fusion proteins and their complements, vectors comprising the nucleic acids, vectors for expressing the fusion proteins in host cells, host cells having episomal or integrated vectors and host cells containing any of the nucleic acids, the recombinant vector, the expression vector and fusion proteins of the present invention in one or more internal cellular compartments.

In In other aspects, the present invention provides for multimeric complexes which contain as several subunits the fusion proteins of the present invention Include inven tion. If they are correct with appropriate soluble Protein partners (eg, β2-microglobulin for fusion proteins, the one class I MHC peptide-presenting Having structural unit) complexed and loaded with peptide, they can self-fluorescent multimeric complexes can be used to To mark T lymphocytes based on their antigen (and MHC) specificity.

In the simplest such aspect, the invention provides a self-fluorescent multimeric protein complex comprising: a plurality of subunits, wherein the subunits have the quaternary formula in the complex of (F) _n , where F is a fusion protein according to the present invention, and n is an integer is greater than 1. If the multimerization domain of the fusion protein is provided by DsRed or a related fusion protein, the complex typically assembles with a tetrameric valency (ie, n = 4).

In a related aspect, the invention further provides a self-fluorescent, multimeric protein complex comprising: a plurality of subunits, wherein the subunits have the quaternary formula in the complex of (F ₁ S ₁ ) _n , where F is a recombinant fusion protein according to the present invention , S is a soluble protein and n is an integer greater than 1.

S is selected from the group consisting of β2-microglobulin, soluble Class II β MHC peptide-presenting Derivatives and class II α-MHC peptide-presenting soluble derivatives. S is β2-microglobulin, when F is a class I α-MHC peptide-presenting Structural unit includes. S is a class II β-MHC peptide-presenting soluble Derivative, when F is a class II α-MHC peptide-presenting Includes structural unit, and S is a class II α-MHC peptide-presenting soluble Derivative when F is a class II β-MHC peptide-presenting Structural unit includes.

The multimeric complex properly assembled with soluble protein (ie, with quaternary (F ₁ S ₁ ) _n stoichiometry) can then be loaded with specific peptide, thereby providing a self-fluorescent reagent useful for detecting label of T lymphocytes based on their antigen specificity.

Thus, in another aspect, the invention provides a self-fluorescent, multimeric protein complex for labeling T lymphocytes based on the specificity of their antigen receptor. The complex of this aspect of the invention comprises a plurality of subunits, wherein the subunits have the quaternary formula in the complex of (F ₁ S ₁ P ₁ ) _n , wherein F, as above, is a recombinant fusion protein of the present invention, S is a soluble Protein as described above, P is a peptide antigen, and n is an integer greater than one.

The Peptide antigen is permeated, as in MHC tetramers and MHC / Ig chimeras the MHC peptide-presenting domains of the linked to the multimeric complex of the present invention: In a self-fluorescent Class I MHC multimer through the α1 and α2 domains of the Class I α MHC peptide-presenting Structural unit of the fusion protein subunit; in a class II multimer through the α1- and β1 domains of MHC peptide-presenting Structural unit and soluble Derivatives of the complex.

Functional includes the self-fluorescent, multimeric protein complex for Labeling of T lymphocytes according to the specificity of their Antigen receptors: means for fluorescing, wherein the Means completely within an amino acid sequence encoded by at least one subunit of the multimeric complex become; and means for binding to a T lymphocyte according to the specificity of its Antigen receptor.

once loaded with a given peptide antigen, the self-fluorescent Complexes of the present invention bind to T lymphocytes which Have antigen receptors specific for the given peptide and MHC peptide-presenting domains of the multimeric complex are specific. Because the peptide antigens and MHC peptide-presenting domains of the Complex of the present invention can both be varied, the self-fluorescent multimeric complexes of the present invention Generally used to an almost unlimited variety on T lymphocytes, based on the antigen (and MHC) specificity of their antigen receptors, to mark fluorescently.

It is therefore another aspect of the present invention to provide methods of using the multimeric complexes of the present invention to target T lymphocytes the specificity of their antigenic receptors to detectably mark (stain).

In a first embodiment the method comprises contacting a T lymphocyte, to be marked with a self-fluorescent multimer A complex of the present invention wherein the complex is peptide antigen and MHC peptide-presenting domains, for which the antigen receptor of the T lymphocyte is specific during one Time and under conditions that are sufficient to be detectable Allow binding of the complex to the T-lymphocyte.

typically, are the T lymphocytes to be labeled within a heterogeneous Sample of cells present, and the target of the label is therein, the antigen-specific T lymphocytes within that population to prove and count often.

Consequently sees the present invention method in another aspect for detecting T lymphocytes specific for a selected antigen, in a sample of cells. The method comprises contacting the sample with a self-fluorescent multimeric complex according to the present invention Invention, wherein the peptide antigen of the complex is the chosen antigen is, and the MHC presentation domains of the Complex ones are those with regard to which the T-lymphocytes, for which the proof desired is restricted be while, and while a time and under conditions that are sufficient to be detectable Bonding of the complex to the chosen one Antigen and MHC-specific T lymphocytes in the sample; and thereafter detecting specific T lymphocytes in the sample the fluorescence of the complex bound thereto. In a related Aspect, the method further comprises counting the so-detected, antigen-specific T lymphocytes.

Depending on The instrument used may be the detection of antigen-specific T lymphocytes coupled directly or indirectly with their sorting, In other aspects of the invention, methods for enriching a Sample and to deplete a sample of T lymphocytes specific for a elected Antigen are to be provided.

in the Generally, the methods of this aspect of the invention include contacting the sample with a self-fluorescent multimeric complex of the present invention, wherein the peptide antigen of the complex the chosen one Antigen, and the MHC presentation domains of the Complex ones are, for which the T-lymphocytes, for which an enrichment or depletion is desired to be restricted be, while a time and under conditions that are sufficient to be detectable Bonding of the complex to the chosen one Antigen and MHC-specific T lymphocytes in the sample. To Binding is labeled T lymphocytes based on fluorescence the complex bound thereto enriched or removed.

such Processes are expediently performed using a fluorescence-activated cell sorter: a Sorting, at least in part, on the fluorescence from the multimers Complex of the present invention, the sample enriches directly from which the cells are removed, and enrich the aliquot, in which the cells are introduced to.

The Multimer complex compositions of the present invention may usefully be provided in the form of kits which facilitate the exercise of Process of the present invention facilitate. Thus, the sees present invention in another aspect, kits, which Self-fluorescent multimeric complexes of the given Invention.

In a series of embodiments, the kits of the present invention include as separate compositions a self-fluorescent (F ₁ S ₁ ) _n multimer of the present invention and an antigenic peptide; As mentioned above, the choice of peptide will depend on the specificity desired for the labeling reagent. In another series of embodiments, the multimer is already pre-loaded with the peptide. Optionally, but advantageously, in any of these series of kits, as separate compositions, any of fluorophore-conjugated antibodies to pan-T or T cell subgrouping antigens, fluorophore-conjugated antibodies specific for T cell activation antigens, may be used. and reagents for lysing red blood cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The mentioned above and other objects and advantages of the present invention considering the following detailed description which in conjunction with the accompanying drawings, become obvious, in which like reference numerals or letters everywhere refer to similar parts, and in which:

1 the construction of a recombinant H2Ld MHC class I-DsRed fusion construct according to the present invention schematically sets;

2 schematically depicts a double-promoter expression vector containing the recombinant H2Ld-DsRed fusion construct according to the present invention;

3 is a photograph of an agarose gel showing the expected 1.5 kD insert for the recombinant H2Ld-DsRed fusion construct after release from NdeI and NotI digested expression vector plasmids;

4 an automatic DNA sequencer track which further confirms the correct DNA sequence at the junction between the expression vector backbone and the H2Ld insert;

5 is a photograph of a stained SDS-PAGE gel showing the induction of the recombinant H2Ld-DsRed fusion protein in transformed E. coli by IPTG;

6 Figure 4 is a photograph of a stained SDS-PAGE gel showing the induction of the recombinant H2Ld-DsRed fusion protein in different transformed E. coli strains by IPTG;

7 is a photograph of a stained SDS-PAGE gel showing the expression of the recombinant H2Ld-DsRed fusion protein in recombinant H2Ld-DsRed baculovirus-infected insect cells;

8th is a conventional microscopic photograph of uninfected Sf9 cells grown in culture for three days;

9 Figure 4 is a photograph of uninfected Sf9 cells grown in culture by fluorescence microscopy using a rhodamine filter grown for three days;

10 Figure 3 is a conventional microscopic photograph of Sf9 cells infected with the recombinant H2Ld-DsRed baculovirus grown in culture for three days after infection;

11 Fig. 10 is a photograph of fluorescence microscopy using a rhodamine filter of recombinant H2Ld-DsRed baculovirus-infected Sf9 cells grown in culture for three days after infection, showing red fluorescence;

12 Figure 3 is a conventional microscopic photograph of Tni cells infected with the recombinant H2Ld-DsRed baculovirus grown in culture for three days after infection; and

13 is a fluorescence microscopy using a rhodamine filter photograph of Tni cells infected with the recombinant H2Ld-DsRed baculovirus, which had been grown in culture for three days after infection, showing red fluorescence.

DETAILED DESCRIPTION OF THE INVENTION

definitions

As used herein have the terms specifically listed below the following definitions. Unless otherwise stated in this section defined, all terms used here have the meaning, which usually by a person skilled in the art to which the invention belongs becomes.

"GFP-like Chromophore "means a self-fluorescent protein residue comprising an 11-stranded β-barrel (β-box) a central α-helix, where the central α-helix a conjugated π resonance system which has two aromatic ring systems and the bridge between includes them. By "self-fluorescent" is meant that the GFP-like Chromophore complete from its amino acid sequence coded and without requirement for co-factor or substrate can fluoresce.

"MHC peptide Structural unit "," MHC peptide-presenting soluble Derivative "and" MHC peptide-presenting domains "all refer to Regions of a major histocompatibility complex protein chain, a transmembrane domain is missing, (and which are therefore "soluble"), and which at the presentation from peptide antigen to a T cell receptor. Of the Term "MHC peptide-presenting Structural unit " used when the soluble MHC range or Section within a fusion protein is included; the Variant "MHC-peptide-presenting soluble Derivative "is used when the protein is expressed unfused; the term "MHC peptide-presenting Domains "becomes generic or genus-specific used in both contexts.

Class I α-MHC peptide-presenting domains include substantially all of the α1, α2 and α3 domains of an MHC class I molecule; By \ "substantially all \" it is meant that deletions thereof and substitutions thereof are permissible only if they confer the ability of the α3 domain to form an intradomain disulfide bond or the ability of α1 and α2 domains to alpha-helices form, which are properly positioned to bind peptide, do not disturb.

Class II α MHC peptide domains shut down essentially all of the α1- and α2 domains of one MHC class II α-chain molecule; with "essentially all "is meant that deletions or substitutions thereof are only permitted if you have the ability the α2 domain, intrachain disulfide bonds train, or ability the α1 domain, one peptide-binding α-helix too form, do not disturb.

Class II β MHC peptide domains shut down essentially all of the β1- and β2 domains of one MHC class II β-chain molecule. With "essentially all "is meant that Deletions thereof or substitutions thereof are only permissible, if you have the ability the β2 domain, intrachain disulfide bonds to form, or the β1-domain, a to form peptide-binding α-helix, do not bother.

fusion proteins

In In a first aspect, the present invention provides a recombinant Fusion protein, which, if properly multimerized and loaded with peptide, without further direct or indirect conjugation An exogenous fluorophore can be used to target T lymphocytes fluorescent labeling based on their antigen and MHC) specificity; The reagents of the present invention may thus be used in all methods be used for which MHC tetramers and MHC / Ig chimeric fusion proteins currently used, including the peptide antigen-specific Activation of T cells.

The Fusion protein of the present invention comprises a GFP-like Chromophore, at least one multimerization domain and an MHC peptide-presenting Structural unit.

One GFP-like Chromophore is a self-fluorescent protein structural unit. By "self-fluorescent" is meant that the GFP-like Chromophore complete from its amino acid sequence and no requirement for a cofactor or substrate can fluoresce.

Structurally includes the GFP-like Chromophore an 11-stranded β-barrel (β-box) with a central α-helix, where the central α-helix has a conjugate π resonance system, which includes two aromatic ring systems and the bridge between them. The π resonance system is produced by autocatalysis under amino acids; the cyclization takes place via an imidazolinone intermediate followed by molecular dehydration Oxygen at the Cα-Cβ bond of a participating tyrosine.

The GFP-like Chromophore may be GFP-like Chromophores selected be in, of course occurring proteins, such as A. victoria GFP (GenBank accession number AAA27721), Renilla reniformis GFP, FP583 (GenBank Accession No. AF168419) (DsRed), FP593 (AF272711), FP483 (AF168420), FP484 (AF168424), FP595 (AF246709), FP486 (AF168421), FP538 (AF168423) and FP506 (AF168422), and only needs so much of that natural Include protein, as needed to maintain the intrinsic fluorescence of the chromophore. Method for determining the minimum domain necessary for fluorescence is required are known in the art; Li et al., Deletions of the Aequorea victoria Green Fluorescent Protein Define the Minimal Domain Required for Fluorescence ", J. Biol. Chem. 272: 28545-28549 (1997).

alternative this may be the GFP-like chromophore made of CFP-like Chromophores selected which are found by those who are found in nature be modified. Typically, such modifications will occur made recombinant production in heterologous expression systems (with or without change in the protein sequence) to stimulate and / or Emission spectra of the native protein change to the purification facilitate, facilitate or as a result of cloning, or it is a random one Consequence of research investigations.

The Method of manipulating such modified GFP-like ones Chromophores and to test these for fluorescence activity, both alone as well as in the form of a part of protein fusions, are in the Field of expertise well known. morning Results of these efforts are summarized in Heim et al., Curr. Biol. 6: 178-182 (1996), incorporated herein by reference is taken; an even more recent review article, with tabular Listing more useful Mutations can be found in Palm et al., Spectral Variants of Green Fluorescent Protein ", in Green Fluorescent Proteins, Conn (Ed.), Methods Enzymol., Vol. 302, Pp. 378-394 (1999), which is incorporated herein by reference. A variety of such modified chromophores are now commercial available and can readily in the fusion proteins of the present invention be used.

To the Example is EGFP ("enhanced GFP"), Cormack et al., Gene 173: 33-38 (1996); U.S. Patents Nos. 6,090,919 and 5,804,387, a red-shifted, optimized for human codon usage Variant of GFP, which for a brighter fluorescence, a higher one Expression in mammalian cells and an excitation spectrum optimized for use in flow cytometers technically changed is. EGFP can be useful in Way a GFP-like Contribute chromophore to the fusion proteins of the present invention. A variety of EGFP vectors, both plasmid and viral Vectors, is commercially available (Clontech Labs, Palo Alto, Calif., USA), including vectors for bacterial Expression, vectors for N-terminal protein fusion expression, Vectors for the expression of C-terminal protein fusions and for bicistronic Expression.

In Towards the other end of the emission spectrum EBFP ("enhanced blue fluorescent protein ", reinforced Blue fluorescent protein) and BFP2 four amino acid substitutions, which are the Emission of green to blue, to increase the brightness of the fluorescence and the solubility of the protein, Heim et al., Curr. Biol. 6: 178-182 (1996); Cormack et al., Gene 173: 33-38 (1996). EBFP is in terms of Expression in mammalian cells whereas BFP2, which retains the original jellyfish codons, is in bacteria can be expressed; as further discussed below, the host cell of production does not affect utility of the resulting fusion protein. The GFP-like chromophores from EBFP and BFP2 can in more useful Included in the fusion proteins of the present invention and vectors containing these blue-shifted variants are from Clontech Labs (Palo Alto, CA).

In contains analogous manner EYFP ("enhanced yellow fluorescent protein "), also available from Clontech Labs, four different amino acid substitutions to EBFP, Ormo et al., Science 273: 1392-1395 (1996), which shift the emission from green to yellow-green. Citrin, an improved yellow fluorescent protein mutant, is described in Heikal et al., Proc. Natl. Acad. Sci. USA 97: 11996-12001 (2000) described. ECFP ("enhanced cyan fluorescent protein ") (Clontech Labs, Palo Alto, Calif., USA) contains six amino acid substitutions, one of which shifts the emission spectrum from green to cyan; home et al., Curr. Biol. 6: 178-182 (1996); Miyawaki et al., Nature 388: 882-887 (1997). The GFP-like Chromophore of each of these GFP variants can be useful in Included in the fusion proteins of the present invention become.

The GFP-like Chromophore can also be used from other modified GFPs be inclusive those described in U.S. Patent Nos. 6 124,128; 6 096 865; 6,090,919; 6,066,476; 6 054 321; 6,027,881; 5,968,750; 5,874,304; 5,804,387; 5,777,079; 5,741,668 and 5,625 048, the same being incorporated herein by reference.

The Fusion proteins of the present invention further include at least one multimerization domain. If multiple multimerization domains exist are, must the domains are not be identical with each other. The multimerization domains can either to effect homomultimerization or heteromultimerization and can to non-covalent or covalent association of the individual subunits contribute.

To the For example, the multimerization domain can usefully be made up of existing, self-dimerizing ones MHC / Ig chimeras are used, which are further described in Dal Porto et al., Proc. Natl. Acad. Sci. USA 90: 6671-6675 (1993); Greten et al., Proc. Natl. Acad. Sci. USA 95: 7568-7573 (1998); Hamad et al. J. Exp. Med. 188: 1633-1640 (1998); Schneck et al., U.S. Patent Nos. 6,015,884 and 6,140,113, the same being incorporated herein by reference. The multimerization structural unit of these chimeras includes one IgG Fc region resulting in disulfide-linked homodimerization in capable.

Other Protein multimerization domains are well known and close for example, leucine zipper domains one.

The Multimerization domain (s) can / can also useful Be provided by the same protein, which is the GFP-like Chromophore provides.

DsRed exists as an inevitable tetramer, even in dilute solution, and has not been observed to monomerize without protein denaturation, Gross et al., Proc. Natl. Acad. Sci. USA 97: 11990-11995 (2000); Wall et al., Nature Structural Biol. 7: 1133-1138 (2000), a property which has been found in the art to be deleterious and interventional ablation or the omission of interventions. In the present invention, this disadvantageous property surprisingly proves useful, allowing DsRed to provide both the GFP-like chromophore and the multimerization domains for the fusion protein of the present invention; with such multimerization domains, the fusion proteins join of the present invention readily to soluble MHC / peptide complexes having the tetrameric valence and thus binding affinity features of known MHC tetramers.

DsRed contains two dissimilar multimerization, which both for its tetramerization seem to be necessary. The smaller one domain shows characteristics typical of many high affinity protein-protein interaction surfaces, and maybe specifically matched to a homo-oligomeric interaction; the larger interface or Junction is dramatic in terms of chemical character different and has features which at oligomerization junctions which require a broader match of substrate specificity seem to be.

The two multimerization domains from DsRed advantageously in the fusion proteins of the present invention Invention in association with the native, GFP-like chromophore of DsRed be used, wherein the excitation (absorption) maximum of about 558 nm, the emission maximum of about 583 nm, a strong resistance against photo-bleaching and strong pH independence of the native DsRed protein to be provided.

alternative can do this the multimerization domains from DsRed with GFP-like Chromophores can be used which are modified from the native DsRed chromophore were. For example, the green-only DsRed mutant K83R (lysine to arginine at the residue) fails 83, numbering as in Baird et al., Proc. Natl. Acad. Sci. USA 97: 11984-11989 (2000)), but to mature to red fluorescence tetramerized nevertheless, and thus becomes useful in the fusion proteins of the present invention, wherein Tetramers are provided with an emission spectrum which easily distinguishable from that of tetramers, which consist of fusions using the native DsRed fluorophore. Conversely, the DsRed mutant has K83M has an emission maximum at 602 nm with relatively little residual green fluorescence, Baird et al., Proc. Natl. Acad. Sci. USA 97: 11984-11989 (2000), and therefore will have another spectral separation of green and yellow emitters Provide fluorophores. Other DsRed chromophore mutants can be light generated, expressed and in terms of spectral and oligomerization characteristics essentially as described in Baird et al., Proc. Natl. Acad. Sci. USA 97: 11984-11989 (2000).

Furthermore although DsRed is only 22% identical in sequence GFP is the total structure of the chromophore removed between these Homologues remarkably conserved. So can the chromophore of A. victoria GFP and its many known variants, including EGFP, EYFP, EBFP and citrin, easily replace the DsRed chromophore, where the spectral properties of the protein are changed while the DsRed multimerization domains are retained. This can be done in an advantageous manner by technical installation of Restriction sites flanking the DsRed chromophore and excision and in-frame replacement of the GFP-like Chromophors from DsRed through cassettes, those for others GFP-like Chromophore coding sequence contained, are suitable in existing DsRed vectors are accomplished.

such DsRed / GFP chimera are therefore suitable for and can be applied advantageously for inclusion in the fusion proteins of the present invention. As further discussed below will, can Fusion proteins of the present invention containing the DsRed multimerization units and dissimilar GFP-like ones Having chromophores, assembled to heteromeric tetramers in which the respective chromophores as donor and acceptor in fluorescence resonance energy transfer ("FRET") reactions take part.

Multimerization domains can also of fluorescent protein homologs provided by DsRed or A. victoria-GFP.

To the For example, Renilla reniformis GFP is believed to be one obligate dimer; Ward, in Green Fluorescent Protein: Properties, Applications, and Protocols, ed. Chalfie & Kain (Wiley, New York) (1998). The Renilla GFP multimerization domain can in more useful Manner used in the fusion proteins of the present invention become the self-organization of complexes with dimeric valency, and thus bond joy characteristics, of existing MHC / Ig chimeras to allow.

Further, Wall et al., Nature Structural Biol. 7: 1133-1138 (2000) reports that oligomerization appears to be a general property of DsRed-like fluorescent proteins such as FP593, FP483, FP484, FP595, FP486, FP538 and FP506. Thus, any of these fluorescent proteins can usefully contribute one or more multimerization domains to the fusion protein of the present invention. As with the use of the DsRed multimerization domains, the fusion protein of the present invention may include the GFP-like chromophore of the fluorescent protein which contributes the multimerization domain (s), [or] a mutation variant thereof, or alternatively may Include GFP-like chromophore of another, homologous, fluorescent protein, or a variant thereof.

The Fusion proteins of the present invention further include an MHC peptide-presenting Structural unit.

The MHC peptide Structure unit is a soluble A region of a major histocompatibility complex protein chain which to participate in the antigen presentation is capable of a T cell receptor, but not itself can be sufficient.

The MHC peptide Structural unit can be derived from MHC class I, MHC class II or non-classical MHC molecules be.

If it is derived from class I, the MHC peptide-presenting Structural unit will be derived from the MHC class I α chain and will include the α1, α2 and α3 domains; permissible deletions from this, or substitutions thereof, the ability of the α3 domain, a Intra-domain disulfide bond to form, or the ability the α1- and α2 domains to form alpha helices, which are properly positioned to bind peptide, do not disturb.

If it is derived from class II, the MHC peptide-presenting Structural unit of the fusion protein of the present invention either from the MHC class II α- or -β-chain be derived; multimeric complexes of the present invention, useful for detecting class II restricted antigen specific T-cell tag, both as discussed further below Fusion proteins with one of class II α as well as one of class II β derived MHC peptide-presenting Include structural unit.

If they are from the class II α chain is derived, the MHC peptide-presenting Structural unit of the fusion protein of the present invention include α1 and α2 domains; permissible deletions from these, or substitutions thereof, may be the ability of the α2 domain, intrachain disulfide bonds to form, or ability the α1 domain, one peptide-binding α-helix to form, do not disturb. If they are from the class II β chain is derived, the MHC-peptide-presenting structural unit of the fusion protein of the present invention include β1 and β2 domains; permissible deletions from this, or substitutions thereof, may be the ability of the β2 domain, intrachain disulfide bonds to form, or the β1-domain, a peptide-binding α-helix train, do not disturb.

If they are derived from a non-classical MHC molecule, the to be included domains depends on, whether the molecule a larger sequence homology to classical class I or class II MHC molecules.

Of the MHC peptide Structural unit will lack the transmembrane region of its respective parent MHC molecule; typically, the MHC peptide-presenting moiety also becomes the cytoplasmic region of the respective MHC molecule is missing.

The MHC peptide Structural unit of the fusion protein of the present invention from the MHC molecules any mammalian or Derived from bird species. For use in multimeric complexes for labeling antigen-specific T cells, the MHC peptide-presenting Structural unit advantageously from the MHC molecules of Species whose T cells are detected should.

Consequently may be the MHC peptide-presenting structural unit of the fusion protein of the present invention in an advantageous manner Way from MHC molecules to humans or other primates, from rodent, in particular Mouse, rat, hamster and guinea pig MHC and rabbit, Derived from bovine, canine, feline and equine MHC. The MHC peptide-presenting Structural unit of the fusion protein of the present invention thus useful from the human class II subunits HLA-DPa, HLA-DPβ, HLA-DQα, HLA-DQβ, HLA-DRα and HLA-DRβ, the human class I subunits HLA-A, HLA-B, HLA-C, murine class II subunits I-Aα, I-Aβ, I-Eα, I-Eβ and murine class I subunits H-2K, H-2D and H-2L are used.

The MHC peptide Structural unit of the fusion protein of the present invention be essentially the same, and may be identical to the ones out there MHC-derived regions of MHC tetramers used in Doherty et al., Annu. Rev. Immunol. 18: 561-92 (2000); Ogg et al., Immunol. Lett. 66 (1-3): 77-80 (1999); Maini et al., Immunol. Today 20 (6): 262-6 (1999); Doherty, Curr. Opin. Microbiol. 1 (4): 419-22 (1998); Reichstetter et al., J. Immunol. 165 (12): 6994-8 (2000); Kwok et al., J. Immunol. 164 (8): 4244-9 (2000); Liu et al., Proc. Natl. Acad. Sci. USA 97 (26): 14596-14601 (2000); Novak et al., J. Clin. Invest. 104 (12): R 63-7 (1999); Crawford et al., Immunity 8: 675-682 (1998); Kozono et al., Nature 369: 151-154 (1994); Altman et al., Science 274: 94 (1996), and Altman et al., U.S. Patent No. 5,635,363 to be discribed.

The MHC peptide-presenting structural unit of the fusion protein of the present invention may be substantially the same, and may be identical, to the MHC-derived portions of MHC / Ig chimeras described in Dal Porto et al., Proc. Natl. Acad. Sci. USA 90: 6671-6675 (1993); Greten et al., Proc. Natl. Acad. Sci. USA 95: 7568-7573 (1998); Hamad et al., J. Exp. Med. 188: 1633-1640 (1998); Schneck et al., U.S. Patent Nos. 6,015,884 and 6,140,113, the disclosures of which are incorporated herein by reference.

Other MHC molecules, which MHC peptide-presenting Structural units can contribute in Sequences of Proteins of Immunological Interest, Kabat et al., (ed.), 5th Ed., U.S. Pat. Dept. of Health and Human Services, Public Health Service, National Institutes of Health (1991), where this disclosure is expressly incorporated herein by reference. and "The Kabat Database of Sequences of Proteins of Immunological Interest ", http://immuno.bme.nwu.edu/, which is further described in Johnson et al., Nucl. Acids Res. 29: 205-206 (2001) will be described.

typically, but not invariable, becomes the MHC-peptide-presenting moiety N-terminal to both the GFP-like Chromophore as well as the multimerization domains of the fusion protein of be located present invention. The relative position of the Chromophors and the multimerization domains in turn become a large part depend on their source.

If for example, the multimerization domain (and possibly also the MHC peptide-presenting Structural unit) from an existing MHC / Ig chimera is the GFP-like one Chromophore favorably attached to the C-terminus of existing ones MHC / Ig chimera fused. As another example, in which the GFP-like chromophore and the multimerization from a single [fluoro] esculting protein, such as DsRed become, the relative positions become by their relative positions in the native protein. The relative positioning of the Multimerization domain (s) and the CFP-like Chromophors can also of cloning considerations depend, being solutions therefor generally within the knowledge of one skilled in the art molecular biology.

In a preferred embodiment includes the fusion protein of the present invention from the N-terminus to the C-terminus, contiguous α1, α2 and α3 domains, taken from a selected MHC class I α subunit what, in the reading frame, essentially the entirety of DsRed follows, which includes both the GFP-like chromophore as well provides two separate multimerization domains. The fusion protein tetramerized spontaneously in solution. The resulting tetramers, after complexation with β2-microglobulin and loading with peptide, and without further labeling, used in all applications be, for which classically class I MHC tetramers and class I MHC / Ig chimeras as useful have proven.

The Fusion protein of the present invention may be useful in Way N-terminal to the MHC presentation unit also include the antigenic peptide itself; this is especially useful in mergers, which is an MHC class II β subunit peptide-presenting Include structural unit; Kozono et al., Nature 369: 151-154 (1994); Liu et al., Proc. Natl. Acad. Sci. USA 97: 14596-14601 (2000), to which expressly herein Reference is made.

The Fusion protein may advantageously also be present either on the N-terminus or C-terminus, include a sequence useful for purification. If it directly adjacent to the MHC-peptide-presenting structural unit is present, this anfusionierte Tag or tag tag is preferred selectively removable.

For example, the fusion protein may include a polyhistidine tag, which may be immobilized by metal affinity chromatography, for example using NiNTA resin (Qiagen Inc., Valencia, CA) or TALON ^™ resin (cobalt immobilized affinity chromatography). Medium, Clontech Labs, Palo Alto, Calif., USA). As another example, the fusion protein may include a chitin-binding tag and self-cutting intein allowing for chitin-based purification with self-removal of the fused tag (IMPACT ^™ system, New England Biolabs, Inc., Beverly, MA, USA ). Alternatively, the fusion protein may include a calmodulin-binding peptide tag permitting purification by calmodulin affinity resin (Stratagene, La Jolla, CA, USA), or a specifically excisable fragment of the biotin carboxylase carrier protein which biotinylates the in vivo purified Protein using an avidin resin followed by tag removal (Promega, Madison, WI, USA).

In alternative embodiments, the fusion protein may also advantageously include a protein spacer which is fused in-frame between the MHC presenting unit and any of the GFP-like chromophore and the multimerization domain (s). The spacer provides a flexible link between the MHC presentation unit and other components of the fusion protein in front. The linker sequence may occur once or as a multiple tandem repeat of the same sequence. Suitable amino acid sequences that provide flexible linkage between the MHC presenting unit and DsRed and techniques for inserting such a spacer into the fusion construct are within the skill of the art in the art.

Nucleic acids, vectors and host cells

The Fusion proteins of the present invention typically become produced by recombinant expression in host cells. Therefore includes the present invention, in further aspects, nucleic acids which encode the fusion proteins described above, vectors which the nucleic acids include vectors for expressing the fusion proteins in host cells, Host cells containing episomal or integrated vectors those for expressing the fusion proteins of the present invention and host cells carrying the fusion proteins of the contained in one or more internal cellular compartments, one.

The nucleic acids The present invention includes sequences encoding the fusion protein or in terms of sequence to those containing the fusion protein encode, are complementary. The nucleic acids (or respective complements thereof) may be the fusion protein as encode a single open reading frame, or can if the encoding nucleic acid for eukaryotic Expression of the fusion protein should be used, the fusion protein in the form of a multitude of exons with intervening introns encode.

The nucleic acids of the present invention may be in the form of DNA, but typically not invariably double-stranded, or in the form of RNA. If the DNA encodes the fusion protein in the form of a plurality of exons, the RNA transcript may exist in both the unspliced introns-containing form and in one or more spliced forms. The nucleic acids of the present invention may also include non-native nucleotides, alternative internucleotide linkages, or both, typically when the nucleic acids are used as probes rather than for expression of the fusion protein. Probes are useful for confirming the transfection of the nucleic acid into host cells and their transcription, although the fluorescence emission itself can often be used as a measure of transfection and expression efficiency, as described in US Pat 11 and 13 will be shown.

The nucleic acids of the present invention technically changed to use codons which are different for expression Types of host cells are optimized, with nucleic acid molecules of different nucleic acid sequence provided that encode the same fusion protein. The nucleic acids of the present invention in isolated and purified form, e.g. In the form of a purified plasmid preparation exist, and can also in the form of nucleic acids which are in a or more chromosomes of a prokaryotic or eukaryotic Host cell are integrated.

As in the examples below, the nucleic acids useful in the present invention Ways to incorporate into vectors the propagation and amplification or duplication the nucleic acids of present invention in one or more types of eukaryotic and prokaryotic host cells, including mammalian cells, in particular Human and rodent cells, insect cells, yeast cells and bacterial Cells, in particular E. coli cells, whereby nucleic acids for the probe construction, for further recombinant procedures, such as mutagenesis, and for cloning be provided in further vectors.

The Vectors of the present invention may be a plasmid, viral or a combination or variant thereof (eg phagmid, cosmid), can one artificial Chromosome (eg BAC, YAC, HAC), or may be any other type of Cloning and / or expression vector, the in the art is known.

vectors can de novo, using techniques well known in the art Techniques as described, inter alia, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press (2000); Ausubel et al. (Ed.), Short Protocols in Molecular Biology: A. Compendium of Methods from Current Protocols in Molecular Biology, 4th Edition, John Wiley & Sons (1999); Jones et al. (Ed.), Vectors: Cloning Applications: Essential Techniques (Essential Techniques Series), John Wiley & Son Ltd. (1998); Cid-Arregui et al. (Ed.), Viral Vectors: Basic Science and Genes Therapy, Eaton Publishing Company / Bio Techniques Books Division (2000), the same being expressly incorporated herein by reference, to be discribed.

alternative can do this the vectors of the present invention in a useful and advantageous manner modified or include relevant areas Vectors currently used to DsRed and / or others fluorescent proteins, MHC tetramers and / or MHC / Ig chimeras and / or to express.

To the An example is a variety of DsRed vectors which are useful as Starting material for constructing the vectors of the present invention Invention are commercially available from Clontech Labs (Palo Alto, CA USA) available. The vector pDsRed is a prokaryotic expression vector, the the native DsRed sequence, flanked by multiple cloning sites; the sequence coding for DsRed can excised by restriction digestion or by PCR amplification be recovered. pDsRed1-1 encodes a DsRed variant with a Valine insertion at position 2, with 144 silent exchanges for optimizing expression in mammalian cells. pDsRed1-N1 allowed easily the mammalian expression mergers to the N-terminus of DsRed; pDsRed1-C1 allows easy the mammalian expression from mergers to the C-terminus from DsRed.

To the Examples are MHC tetramer expression vectors used as starting material useful for constructing the vectors of the present invention, described in Doherty et al., Annu. Rev. Immunol. 18: 561-92 (2000); Ogg et al., Immunol. Lett. 66 (1-3): 77-80 (1999); Maini et al. Immunol. Today 20 (6): 262-6 (1999); Doherty, Curr. Opin. Microbiol. 1 (4): 419-22 (1998); Reichstetter et al .; J. Immunol. 165 (12): 6994-8 (2000); Kwok et al., J. Immunol. 164 (8): 4244-9 (2000); Liu et al., Proc. Natl. Acad. Sci. USA 97 (26): 14596-14601 (2000); Novak et al., J. Clin. Invest. 104 (12): R 63-7 (1999); Crawford et al. Immunity 8: 675-682 (1998); Kozono et al., Nature 369: 151-154 (1994); Altman et al., Science 274: 94 (1996) and Altman et al., U.S. Patent No. 5,635,363, the disclosures of which are expressly incorporated by reference is taken.

To the Examples are vectors for the expression of MHC / Ig chimeras, which useful as starting material for constructing the vectors of the present invention Invention are described further in Dal Porto et al., Proc. Natl. Acad. Sci. USA 90: 6671-6675 (1993); Greten et al., Proc. Natl. Acad. Sci. USA 95: 7568-7573 (1998); Hamad et al., J. Exp. Med. 188: 1633-1640 (1998); Schneck et al., U.S. patents Nos. 6,015,884 and 6,140,113, the disclosures of which are hereby incorporated by reference expressly Reference is made described.

Especially useful among the vectors of the present invention are those which for expression of the fusion proteins of the present invention in Host cells can be used. details Expression vector construction is well known in the art and must not summarized here again. Generally speaking, however, conclude the expression vectors of the present invention are those which replicate episomally within the host cell, and those that integrated at one or more sites of the host cell genome, one. Expression of the fusion protein from the vector may be be constitutive or inducible, and the expressed fusion protein can be retained within the cell or be secreted.

The present invention includes host cells containing the nucleic acids, typically the vectors, including Expression vectors of the present invention either episomally or integrated at one or more sites in the host cell chromosome contain. If integrated, the expression vector is in a useful way in multiple copies, causing the increased copy number can by amplification techniques, which are well known in the art See, for example, Axel et al., U.S. Patent No. 4,399,216, herein incorporated by reference expressly Is referred). If the host cells expression vectors of the typically include the host cells Further, the fusion proteins of the present invention in one or several cellular Compartments.

The Host cells of the present invention may contain a variety of vectors, including Expression vectors of the present invention; the variety Vectors can each have a variety of different fusion proteins encode, whereby expression of heteromeric multimers within the cell is allowed. As will be further described, such heteromeric multimers in useful Way to include fusion proteins, but the identical MHC antigen presenting unit and multimerizing domains different GFP-like ones Have chromophores.

to Propagation of nucleic acids useful in the present invention Host cells close bacterial Cells, especially E. coli cells, and eukaryotic cells.

For the recombinant Production of the fusion proteins of the present invention useful host cells shut down all types of cells that are typically recombinant Expression can be used, including both prokaryotic as well as eukaryotic cells, the latter human, Include rodent, insect and yeast cells.

Insect cells, such as Sf9 and Tni cells, provide certain advantages for the expression of the fusion proteins of the present invention, including the high yield (see e.g. 11 and 13 ) and a likely low loading of endogenous peptide, which is erroneously loaded onto the nascent multimers during synthesis. Rodent myeloma cells offer in particular There are certain advantages to expression when the fusion protein includes most or all of an Ig / MHC chimera fused to a GFP-like chromophore; Among such advantages, the simultaneous production of Ig light chain is remarkable.

If the fusion protein to be expressed represents an MHC peptide Including structural unit of a class I MHC molecule, the host cells of the present Invention further useful Way and at the same time β2-microglobulin, either endogenous or over expressing the expression of a second recombinant construct.

If the expressed fusion protein for multimerization in the intracellular milieu is able to close the host cells typically further include such multimers.

Multimeric complexes

In In another aspect, the present invention provides multimeric complexes which, in the form of several subunits, fusion proteins of the present Invention. When appropriate with appropriate soluble protein partners (e.g. B. β2-microglobulin for fusion proteins with a class I MHC peptide-presenting Structural unit) are complexed and loaded with peptide, these can self-fluorescent multimeric complexes can be used to To mark T lymphocytes based on their antigen (and MHC) specificity.

In its simplest form, the multimeric complex of the present invention comprises a multimer of the fusion proteins of the present invention resulting in the complex of a quaternary formula of (F) _n , wherein F is a fusion protein as described above, and n is an integer is greater than 1.

The Multimerization is performed by the multimerization domains of the Fusion protein driven and mediated. Where the multimerization domain (s) of the Fusi onsprotein subunits from DsRed, variants thereof or related fluorescent proteins are used, the Fusion protein subunits typically predominantly to tetramers (n = 4) put together with a possible Formation of weakly associated octamers (n = 8). If the multimerization domain (s) of the Fusion protein subunits instead of Renilla-GFP or from IgG Fc regions, the fusion protein subunits will become typically combine predominantly into dimers (n = 2).

If the fusion protein subunits in eukaryotic cells, such as Rodent myeloma cells or Insect cells, expressed, become the fusion protein subunits typically within the cell to complexes of expected valency put together by itself. When the fusion protein subunits instead expressed in prokaryotic cells such as E. coli Typically, the subunits will only become denatured (eg in 8 M urea) and renaturing together, like it is well known in the MHC tetramer art.

Of the resulting multimeric complex is self-fluorescent due to the presence of GFP-like Chromophores in each of the participating subunits. The spectral features of the complex are not only of the spectral properties the individual GFP-like compounds present within the complex Given chromophores but also by their collective interaction.

A a particularly important interaction among GFP-like chromophores is the Fluorescence Resonance Energy Transfer ("FRET").

Of the The multimeric complex of the present invention may include fusion protein subunits different CFP-like Include chromophores, as long as the fusion protein subunits remain able to control their respective multimerization domains to multimerize. Typically, but not invariable, will be the fusion protein subunits of such heteromultimeric complexes all the same MHC peptide-presenting moieties exhibit. If the different GFP-like chromophores have a suitable spectral overlap can exhibit their closeness and relative angular displacement within the multimeric complex a fluorescence resonance energy transfer between the different GFP-like chromophores allow.

For example, it is known that the DsRed chromophore can be used as an acceptor in a Fluorescence Resonance Energy Transfer (FRET) pair with Enhanced Green Fluorescent Protein (EGFP) or Yellow Fluorescent Protein (YFG) mutants; Heikal et al., Proc. Natl. Acad. Sci. USA 97: 11996-12001 (2000). In addition, fluorescence resonance energy transfer among monomers in the native DsRed tetramer has been reported. Thus, where the multimeric complex of the present invention includes both a fusion protein subunit with a GFP-like chromophore of DsRed and a fusion protein subunit with a GFP-like chromophore of EGFP, the spectral properties of the complex may include fluorescence resonance energy transfer between EGFP and DsRed chromophores reflect what allows the complex to be lighter at the EGFP absorption maximum is stimulated than would have been possible with a homotetramer, which has only the DsRed chromophores.

As noted above, fluorescence resonance energy transfer can occur between and under CFP-like Chromophores take place in the subunits of the multimers Complexes of the present invention are present. A FRET can about that out and in more useful Way between the GFP-like Chromophore of one or more of the subunits of the complex and a fluorophore associated therewith.

techniques for associating a fluorophore with a subunit of the multimeric Complex so that the FRET is supported - by conjugating, linking or Add of the fluorophore to the subunit, or otherwise modify a subunit with the fluorophore - are within the knowledge of the skilled person in the field.

Depending on the absorption and emission spectra of the fluorophore, which associated with a particular type of GFP-like chromophore is either the fluorophore or the GFP-like chromophore as the Fluorescence energy donor or acceptor serve.

Fluorophores useful as FRET donors or acceptors when associated with one or more subunits of the multimeric complex of the present invention include the following: cyanine 3 (e.g., Cy3, Cy3B); Cyanine 5 (e.g., Cy5, Cy5Q); Cyanine 5.5; Cyanine 7 (e.g., Cy7Q); SYBR ^® Green (Molecular Probes); Fluorescein and fluorescein derivatives (e.g., 6-carboxyfluorescein); Rhodamine and rhodamine derivatives (eg, dichlororrhodamine, dR110, dR6G, dTAMRA, dROX); Allophycocyanin and allophycocyanin derivatives (e.g., GT5 APC); R-phycoerythrin; B-phycoerythrin, Y-phycoerythrin; R-phycocyanin; C-phycocyanin; Texas ^Red® ; and proteinaceous fluorophores, e.g. B. PerCP.

According to one preferred embodiment is used the GFP-like Chromophore as the FRET donor, and the fluorophore serves as the FRET acceptor. In this way, stimulating light energy of the appropriate wavelength of the CFP-like Absorbs chromophore and is transferred intramolecularly to the fluorophore, which then fluoresces the energy at a different, longer wavelength emitted.

These Arrangement can increase the speed of fluorescent light emission in more useful Speed up the way.

The Rate of fluorescence decay of some GFP-like Fluorophores after light stimulation is relatively slow. For example, this decays excited or energized DsRed chromophore by fluorescence emission via a Duration of microseconds to its ground state. In contrast In addition the intramolecular energy transfer takes place by means of FRET a CFP-like Chromophore, such as DsRed, and an associated fluorophore typically in nanoseconds. As a result, the fluorescence emission be faster than the fluorescence emission by the fluorophore through the CFP-like Fluorophore. The effect of this difference in speed the fluorescence energy emission is that within a short predetermined time period, the fluorescent light emission through the fluorophore, which is coupled to the GFP-like chromophore via FRET is, is bigger, and the signal appears brighter. In contrast, within the same time the fluorescent light emission by the GFP-like chromophore, which not supported by FRET and a fluorophore, lower, and that Signal appears weaker.

The increase the rate of fluorescence emission of GFP-like chromophores, such as such as by the FRET-mediated mechanism described above, may be particularly advantageous be in the application of flow cytometry. The FRET-mediated Acceleration of fluorescence emission by associating a fluorophore with a CFP-like Chromophore also becomes desirable in any other application be in which the signal acquisition must be done quickly or Signal must be maximized.

in the Generally, the appropriate selection of GFP-like chromophores will be in which fusion protein subunits are to be included that multimerizes in the complex (and optionally FRET donors and acceptors attached thereto conjugate), the fine tuning of the spectral characteristics of the Allow complex. An important application of such a vote consists of the absorption and emission characteristics of the Complex to the excitation and detection parameters of the analyzer, typically of a flow cytometer. Conversely, as below further discussed will, can Lasers, filter sets and detectors are chosen, which the absorption and emission characteristics of the multimers Complex correspond.

For the detectable labeling of antigen-specific T lymphocytes, the multimeric complex of the present invention also becomes soluble Include protein partners and peptides. However, the (F) _n complexes described above are useful intermediates in the preparation of these labeling reagents and will usefully be sold to end users who wish to complete the formation of the complex themselves.

Thus, in another aspect, the invention includes self-fluorescent, multimeric protein complexes comprising a plurality of subunits, said subunits having the quaternary formula in the complex of (F ₁ S ₁ ) _n , wherein F, as described above, is a recombinant Fusion protein of the present invention, S is a soluble protein, and n is an integer greater than one.

S is chosen from the group, which consists of β2-microglobulin, Class II β MHC peptide-presenting soluble Derivatives and class II α-MHC peptide-presenting soluble Derivatives is: S is β2 microglobulin, when F is a class I α-MHC peptide-presenting Includes structural unit; S is a class II β-MHC peptide-presenting soluble Derivative, when F is a class II α-MHC peptide-presenting Includes structural unit; and S is a class II α-MHC peptide-presenting soluble Derivative when F is a class II β-MHC peptide-presenting Structural unit includes.

typically, is the soluble Protein derived from the same taxonomic species, which the MHC peptide-presenting Provides structural unit of the fusion protein in the complex. If the fusion protein includes, for example, murine MHC class I α domains the soluble β2-microglobulin used in the multimeric complex is a murine β2-microglobulin be. For example, when the fusion protein includes human HLA DQα domains the MHC peptide-presenting soluble derivative included within the complex human class II β domains, such as such as HLA-DQβ domains.

The soluble protein can be coexpressed with the fusion protein subunits in a single host cell, facilitating the self-organization of the (F ₁ S ₁ ) _n complex within the cell, or can become a previously assembled complex containing the quaternary formula F _n , be added. When the fusion protein subunits of the multimeric complex include a class II MHC peptide-presenting moiety, the soluble class II MHC peptide-presenting derivative will most typically be co-expressed therewith, especially when the expression of the fusion protein is effected in eukaryotic cells ; When the fusion protein subunits of the multimeric complex include a class I α-MHC peptide-presenting moiety, the soluble β2-microglobulin protein can be readily co-expressed with it or added after assembly of the (F) _n multimer. Coexpression of β2-microglobulin in eukaryotic cells may result from either expression from a recombinant construct or, depending on the choice of host cell, from endogenous expression by the host cell itself.

Of the multimeric complex correctly assembled with soluble protein, can then be loaded with specific peptide, creating a self-fluorescent Reagent is provided for the detectable label (and in other methods the activation) of T lymphocytes based on their antigenic specificity useful is.

It is understood that the complex described above - ie, including fusion protein and soluble protein subunits, but without peptide antigen - can be loaded with a tremendous variety of peptide antigens, each peptide conferring its own specificity to the completed labeling reagent. The (F ₁ S ₁ ) _n complex described above is therefore extremely useful in the preparation of labeling reagents; and often the self-fluorescent complexes of the present invention are usefully sold to the end user in a form that allows the end user to load the complex with the peptide antigen of his or her choice.

Loaded with peptide antigen, the multimeric complex is directly useful to label T lymphocytes based on the antigen (and MHC) specificity of their T cell receptors. Thus, in another aspect, the invention provides a self-fluorescent multimeric protein complex for labeling T lymphocytes, said complex comprising a plurality of subunits, said subunits having the quaternary formula in complex of (F ₁ S ₁ P ₁ ) _n in which F, as above, is a recombinant fusion protein of the present invention, S is a soluble protein as described above, P is a peptide antigen, and n is an integer greater than one.

The peptide antigen, as in MHC tetramers and MHC / Ig chimeras, will be bound by the MHC peptide-presenting domains of the multimeric complex of the present invention: in a self-fluorescent Class I MHC multimer through the α1 and α2 domains the class I α-MHC peptide-presenting structural unit of the fusion protein subunit; in a class II multimer through the α1 and β1 domains of the MHC peptide-presenting structural unit and soluble derivatives of the complex.

If the complex of the present invention is a class I multimer, For example, the peptide antigen will typically be no less than about a length 5, more typically not less than about 6, more typically not less than about 7, often not less than about 8 or 9 amino acids, and typically a length not more than about 15, typically not more than about 14, more typically not more than about 13, 12 or 11 and often not more than about 10 or 9 amino acids exhibit.

As it is well known in the field of immunology, the antigenic peptides have a broader length range, when to MHC class II are complexed. For loading class II multimers, the Peptides therefore typically have a length of not less than about 8, typically not less than about 9, 10, 11 or 12 amino acids, and typically become a length not more than about 30, typically not more than about 25, typically not more than about 19, 18, 17 or 16 amino acids, often one length of not more than about 12 amino acids, exhibit. For Class II multimers can use the Furthermore, peptides covalently bind to the N-terminus as mentioned above the MHC peptide-presenting structural unit or a soluble one Derivatives, Kozono et al., Nature 369: 151-154 (1994), Liu et al., Proc. Natl. Acad. Sci. USA 97: 14596-14601 (2000), rather than non-covalently to be associated.

peptides typically become substantially pure and uniform Sequence, as they are advantageously prepared by chemical synthesis can, what is possibly a further purification, such. B. by HPLC, followed. For some However, applications can have a collection of peptides - e.g. B. a collection of peptides useful for vaccinating against CMV, as described in WO 00/75180, the disclosure of which is hereby incorporated by reference Reference is made - used be present to the self-fluorescent multimers of the present To load invention. In this latter case becomes a population generated by multimers that collectively mark T lymphocytes, which recognize any of the loading peptides. For such ge Purposes can the peptides advantageously by endoproteolytic digestion of a native protein, such as a viral protein.

peptides can non-native amino acids and include non-native bonds, if such non-native ones amino acids and / or non-native linkages the ability of the self-fluorescent complex, T lymphocytes mark, do not disturb. For example, non-native Amino acids and / or Bindings are used to determine the T cell response to the immunization with a protein or peptide containing such non-native amino acids or Includes bonds, or to detect T lymphocytes responsible for the peptide in are specific to its native form if the non-native amino acids and / or Bindings do not affect the three-dimensional structure of the peptide.

If the multimeric complexes of the present invention by recombinant Expression in eukaryotic cells, especially mammalian cells the multimer can be mistaken include endogenous peptides of the host cell. Although an expression in insect cells the extent of loading may decrease with endogenous peptide, nonetheless certain percentage of peptide binding sites before the intended one Loading be occupied. In such cases, the peptide loading becomes of the multimeric complexes include some level of peptide exchange. The exchange can at higher Temperatures, like 37 ° C, and by using higher Concentrations of loading peptide are facilitated. if the Concentration of the peptide used for loading is high, and indeed at any peptide concentration, dialysis may be optional completion of loading to reduce the concentration of unbound peptide.

The sequence of the loading peptide is dictated by the application for which the labeling reagent is to be used. For example, to detect the antigen-specific CD8 ⁺ T cell response to viral infection or viral vaccination, a peptide sequence derived from viral protein is used to load a self-fluorescent Class I multimer; To detect the presence of autoimmune T lymphocytes associated with multiple sclerosis, a peptide derived from myelin basic protein (myelin basic protein) can be used to load a self-fluorescent class I or class II multimer ,

As mentioned, peptides are from viruses such as HIV (human immunodeficiency virus), SIV (monkey immunodeficiency virus), CMV (cytomegalovirus), HBV (hepatitis B virus), HCV (hepatitis C virus), HSV (Herpes simplex virus), EBV (Epstein-Barr virus) and HPV (human papilloma virus), especially useful. Also useful are bacterial peptides, such as mycobacterial peptides, including TB. Another class of peptides that are used to advantage are the peptides derived from cancer-related antigens In particular, cancer antigens are used in potential cancer vaccines, especially melanoma, prostate cancer and breast cancer vaccines.

In Peptides used in the multimeric complexes of the present invention can also based on their mimicry of non-peptide structures, like carbohydrates, selected become; Luo et al., J. Biol. Chem. 275 (21): 16146-54 (2000); O et al., Biochem. Biophys. Res. Commun. 5; 268 (1): 106-11 (2000); Grothaus et al. Vaccine 18 (13): 1253-63 (2000); and Phalipon et al., Eur. J. Immunol. 27 (10): 2620-5 (1997).

The multimeric complexes of the present invention, with or without associated peptide (ie either with the quaternary formulas (F ₁ S ₁ ) n or (F ₁ S ₁ P ₁ ) _n ), may usefully be used as an aqueous composition, typically including buffers, salts and / or stabilizers, e.g. Phosphate buffered saline with gelatin and 0.1% sodium azide, and it is therefore another aspect of the present invention to provide such compositions. Alternatively, the complexes can be provided as nonaqueous, lyophilized compositions.

application process

As soon as they are loaded with a given peptide antigen, the autofluorescent complexes of the present invention bind to T lymphocytes, which possess antigenic receptors specific to the given Peptide and the MHC peptide-presenting domains of the multimeric complex. Because the peptide antigens and MHC peptide-presenting domains the complexes of the present invention can both be varied, the self-fluorescent multimeric complexes of the present invention Generally used to an almost unlimited variety of T lymphocytes based on the antigen (and MHC) specificity of theirs Fluorescently label antigen receptors.

It is therefore another aspect of the present invention, method to provide for the use of the multimeric complexes of the present invention, detectable for T lymphocytes based on the specificity of their antigen receptors to mark (color).

In a first embodiment the method comprises contacting a T lymphocyte, which with a self-fluorescent multimeric complex of to mark the present invention, wherein the complex a Peptide antigen and MHC peptide-presenting domains has, for which is the T lymphocyte antigen receptor specific, and while while a time and under conditions that are sufficient to detect the detectable bond of the complex at the T lymphocytes.

in the The present context is said to be specific to a T lymphocyte for a To be peptide antigen if the affinity of its antigen receptor (TCR) for the Peptide antigen in the MHC context of the complex is sufficiently high, to give the complex as a whole attachment to the T lymphocyte, which is sufficient to detect a detectable binding of the complex to achieve TCRs on the lymphocyte surface.

Become frequent the peptide antigen (s), for which the T lymphocyte according to this definition assert specific is also capable of a Zy tokin expression by the To stimulate T lymphocytes; such a functional answer However, it is not necessary because of the usefulness of multimeric complexes of the present invention, as for MHC tetramers the labeling and identification of functionally responsive T lymphocytes limited is. Furthermore, with this definition, it is for a single T lymphocyte that has a single αβ or γδ TCR species on its surface not impossible, assertedly for one Variety of (typically closely-related) peptide antigens to be. In such cases Typically, the TCR will have a greater affinity for one the peptides as for the others own.

conditions and times for Such labels are suitable to those skilled in the art for the coloring of T lymphocytes with MHC tetramers or MHC / Ig fusions conveniently be adjusted.

For example, Altman et al., Science 274: 94-96 (1996), stained 200,000 cytotoxic lymphocytes with MHC tetramers by incubation at 4 ° C for one hour at a tetramer concentration of approximately 0.5 mg / ml; The NIAID "Tetramer Facility" (http://www.niaid.nih.gov/reposit/tetramer/genguide.htm) currently recommends staining at 4 ° C, room temperature and 37 ° C for 15-60 minutes each to optimize the signal-to-background ratio, using decreasing incubation times for higher temperatures; Greten et al., Proc. Natl. Acad. Sci. USA 95: 7568-7573 (1998) stain 1 x 10 ⁶ peripheral blood mononuclear cells at 4 ° with 3 μg of MHC class I MHC / Ig chimera.

Thus, T lymphocytes can be conveniently labeled with the self-fluorescent multimeric complexes in the methods of the present invention using at least about 0.1 μg, typically at least about 0.25 μg, more typically about 1 μg, 2 μg, 3 μg, 4 μg, or even at least about 5 μg of multimer, by about 10 ⁴ , 10 ⁵ , 10 ⁶ or even 10 ⁷ peripheral blood mononuclear cells using a 15-60 minute incubation at a temperature between 4 ° C and 37 ° C.

outgoing Of these rough exemplary guidelines, those skilled in the art will become aware the field of T lymphocyte labeling using MHC tetramers, MHC / Ig fusions and fluorophore-conjugated antibodies readily in the Be able to determine optimal marking conditions. Variables, which the amount of multimeric reagent to use and the temperature and Duration of staining influence, close those related to T lymphocytes - the number of T lymphocytes in the sample specific for the peptide antigen (and the MHC) of the complex, the total number of cells in the sample, the Form of the cellular Sample (eg whole blood, whole blood after lysis of the red blood cells, Ficoll-purified Fraction of peripheral blood mononuclear cells (PBMC)) - and those which are related to the multimeric complex, including the stoichiometry and the molecular weight of the label complex, the identity of the peptide antigen and the selection of MHC alleles which are included in the complex are a.

Around To optimize labeling conditions can be labeling reactions readily using parallel aliquots of those to be labeled Cell sample performed be a single species of multimeric complex and varying Marking conditions (eg temperature, duration, complex concentration, Cell number, cell concentration, cell purity). negative controls can Labeling reactions without the use of multimer, using of multimer without peptide and / or using multimer, the MHC peptide-presenting domains and / or Contains peptide antigens, which are not recognized by T lymphocytes in the cell sample. The effectiveness The labeling can readily be performed by flow cytometric counting of each aliquot T lymphocytes in the sample containing the fluorescent complex bind, be determined. As in the field of flow cytometry well known, can the labeled cells are washed before flow cytometry, to unbonded complex from the cells and from the medium too remove. All these optimization techniques and procedures are routine matters and will be of the professional in the field flow cytometry routinely performed.

typically, are the T lymphocytes to be labeled within a heterogeneous Sample of cells present, and the target of the label is therein, the antigen-specific T lymphocytes within that population prove and often also to count.

Consequently In another aspect, the present invention provides methods for detecting T-lymphocytes specific for a selected antigen, ready in a sample of cells. The method comprises contacting the sample with a self-fluorescent multimeric complex according to the present invention Invention, wherein the peptide antigen of the complex is the chosen antigen is, and the MHC presentation domains of the complex those are those with regard to which the T-lymphocytes for which the Proof is desired, be restricted, for a while and under Conditions sufficient to detect the detectable binding of the complex T lymphocytes in the sample specific for the antigen and MHC chosen are to be allowed; and thereafter detecting specific T lymphocytes in the Sample by the fluorescence of the complex bound thereto.

Detection of cell-bound fluorescence is typically performed using a flow cytometer, such as a FACSVantage ^™ , FACS-Vantage ^™ SE or FACSCalibur ^™ flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). The lasers chosen for the excitation are determined by the absorption spectrum of the multimeric complex and any additional fluorophores for which simultaneous detection is desired in the sample. For example, if the multimeric complex has the spectral characteristics of native DsRed - e.g. For example, a homotetramer in which the fusion protein subunits all have the native GFP-like chromophore of DsRed - a standard argon ion laser with 488 nm line can be used for the excitation. For detection, the filter sets and detector types are chosen according to the emission spectrum of the multimeric complex and any additional fluorophores whose detection is desired in the sample. For example, if the multimeric complex has the spectral characteristics of native DsRed, with an emission maximum at about 583 nm, fluorescence emission from the complex can be detected in the FL2 channel using a PE setup.

alternative this can be done by using cell-bound complex fluorescence a microvolume fluorimeter such as the IMAGN 2000 (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA).

applications of microvolume fluorimetry on the characterization of blood cells and conditions for that are reported, inter alia, in Seghatchian et al., Transfus. Sci. 22 (1-2): 77-9 (2000); Glencross et al., Clin. Lab. Haematol. 21 (6): 391-5 (1999), and Read et al., J. Hematother. 6 (4): 291-301 (1997).

alternative this can be cell-bound complex fluorescence using a Laser Scanning Cytometer (Compucyte Corp., Cambridge, MA, USA) be detected.

The Cell-bound fluorescence of the multimeric complex may also be directly on a microscope slide using conditions be detected, essentially as described in Skinner et al., "Cutting edge: In situ tetramer staining of antigen-specific T cells in tissues ", J. Immunol (2): 613-7 (2000).

The T lymphocyte-containing sample may be a whole blood sample, typically a sample of peripheral venous blood taken directly into an anticoagulant sample tube (e.g., EDTA-containing or heparin-containing Vacutainer ^™ tube, Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA).

Advantageously, the sample containing T lymphocytes may also be a whole blood sample that has been treated with a red blood cell lysing agent (RBC) prior to detection, as described, inter alia, in Chang et al., U.S. Patent Nos. 4,902,613 and 4,654,312. Lysing agents are well known in the art and are commercially available from a number of manufacturers (FACS ^™ Lysing Solution, Becton Dickinson Immunocytometry Systems, San Jose, Calif., USA; Cal-Lysis ^™ Lysing Solution, Caltag Labs, Burlingame, CA, USA; No-Wash Lysing Solution, Beckman Coulter, Inc., Fullerton, CA). The sample may optionally be washed after RBC lysis and before detection.

The sample within which the detection of T-lymphocytes is desired according to the methods of the present invention may further be a fraction of peripheral blood, advantageously a fraction of mononuclear cells (PBMC). PBMCs may be prepared according to any of the well-known techniques known in the art, among which centrifugation through a density medium such as Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and direct centrifugation into a specially designed cell preparation bleed Tubes (e.g., Vacutainer ^™ CPT ^™ Cell Preparation Tube, Becton Dickinson, Franklin Lakes, NJ, USA).

The Sample, within the detection of T lymphocytes according to the procedures of the present invention can be advantageously also a sample, which in terms of Enriched T lymphocytes. For example, it may be at the Sample around a sample of cultured lymphocytes (as from a clonal cell line or multiclonal culture), lymphocytes, the were extracted or eluted from a lymphocytic tissue Infiltrate (e.g., tumor infiltrating lymphocytes, the extracted from a tumor biopsy and optionally propagated in culture ), lymphocytes taken from lymphatic vessels or thymus, or lymphocytes activated after at least a first round of fluorescence Cell sorting were obtained, act.

In another embodiment The method further comprises counting the thus detected antigen-specific T lymphocytes. Counting may be appropriate in the form of a total cell count, as a percentage of antigen-specific T lymphocytes among the Assayed cells (either total, mononuclear or T lymphocytic) or as a percentage of antigen-specific lymphocytes in one T cell subgroup expressed become.

For many these dimensions it is necessary in addition the total number of T lymphocytes in the sample as a whole or the total number of T lymphocytes one particular subgroup within the sample as a whole.

Consequently includes the method of the present invention in other embodiments further contacting the sample with at least one fluorophore-conjugated Antibody, wherein the antibody selected from the group consisting of pan-T antibodies and T-cell subgrouping antibodies, and then detecting fluorescence, simultaneously from the multimeric fluorescent complex and of the fluorophore-conjugated antibodies. By "pan-T antibody" is meant an antibody which a surface marker or recognize an epitope that is at all or essentially all T lymphocytes is present. By "T cell subgrouping antibody" is meant an antibody which binds to a surface marker, which is present on less than all T lymphocytes.

Antibodies useful in this embodiment include antibodies specific for CD3, CD4, CD8, CD45RO, CD45RA and CD27. It is understood that the antibodies will typically be specific to the marker as expressed by the taxonomic species (human, mouse, rat, etc.) for whose T lymphocytes the detection is made, or to be cross-reactive.

As it is well known in the field of flow cytometry, For example, the fluorescence emission from the pan-T and / or T-lymphocyte subgrouping antibodies may be for each or anything under "triggering" data collection, "live gating" or "gating" from previously detected Data are applied.

In it is often advantageous to the processes of the present invention a big Number of events, since in many samples antigen-specific T lymphocytes do not often occur. About that In addition, it is sometimes possible the signal-to-background ratio to detect such rare antigen-specific T-cell events by "triggering" or "gating" with respect to Fluorescence of antibodies, the for T lymphocyte activation antigens are specific.

Therefore For example, in another embodiment, the method further comprises contacting the sample with at least one fluorophore - conjugated antibody, the for a T cell activation antigen, and then detecting the fluorescence simultaneously from the multimeric fluorescent Complex and from the fluorophore-conjugated antibodies. The antibodies can be in more useful Way for be an activation antigen, consisting of the group consisting from CD69, CD25, CD71 and MHC class II (for labeling human T lymphocytes advantageous HLA-DR), selected is.

Favorable The manner in which these embodiments antibodies used in the methods of the present invention Precursor conjugated directly to a fluorophore, typically a fluorophore, whose emission is flow cytometrically distinguishable from that of the self-fluorescent multimeric T-cell labeling complex and of those of other fluorophores which occur simultaneously in the Procedure can be used. Fluorophores may be useful from the group chosen made from fluorescein isothiocyanate (FITC), Phycoerythrin (PE), peridinin-chlorophyll protein (PerCP), allophycocyanin (APC), Texas Red, Alexa Fluor 488 (Molecular Probes, Inc., Eugene Or.) And the tandem fluorophores PerCP-Cy5.5, PE-Cy5, PE-Cy7 and APC-Cy7 exists. Antibodies can also in more useful Be conjugated to biotin, giving a second-stage detection using fluorophore-labeled streptavidin.

The methods of the present invention for detecting and counting antigen-specific T lymphocytes can be used for the same purposes as methods for detecting and counting prior art antigen-specific T lymphocytes, including methods based on surface labeling of TCR (use of MHC tetramers and MHC / Ig chimeras), as well as methods based on detecting functional responses of antigen-specific T cells (limiting dilution assay, enzyme-linked immunospot assay, and flow cytometric detection of intracellular cytokine expression; Waldrop et al., J. Clin. Invest 99 (7): 1739-50 (1997)). The method can thus z. B. can be used to evaluate CD4 ⁺ and CD8 ⁺ T cell responses to infection, vaccines and autoimmunity.

Depending on The instrument used may be the detection of antigen - specific T lymphocytes coupled directly or indirectly with their sorting which, in other aspects of the invention, provide methods for Enriching a sample and depleting a sample of T lymphocytes, specific for a chosen one Antigen are to be provided.

in the Generally, these methods involve contacting the Sample with a self-fluorescent multimeric complex of the present Invention, wherein the peptide antigen of the complex is the chosen antigen is and the MHC presentation domains of the Complex ones are those with regard to which the T-lymphocytes, whose enrichment or removal is desired to be restricted be, while a time and under conditions that are sufficient to detect the detectable bond of the complex of T lymphocytes in the sample, which for the chosen one Antigen and MHC are specific to permit. After the binding are labeled T lymphocytes based on the fluorescence of the complex, enriched or removed.

such Procedures are conventional performed using a fluorescence-activated cell sorter: The Sorting, which is based at least in part on the fluorescence from the based multimeric complex of the present invention enriches the sample directly from which the cells are removed and enriched the aliquot into which the cells are introduced.

It is possible, however to use the multimers of the present invention to sample To enrich or deplete T lymphocytes of particular antigenic specificity, using procedures other than fluorescence-activated cell sorting be applied.

For example, T lymphocytes specifically stained with the multimer can be magnetically, rather than fluorometrically, by further conjugation of the TCR-bound complex to a superparamag to be separated from a net particle. This can be z. Example, using an antibody which is conjugated to a magnetic particle, and which is specific for an epitope of the fusion protein (for example, if DsRed contributes the GFP-like chromophore and / or Mulimerisierungsdomänen, the antibody of the DsRed-specific Antibody commercially available from Clontech Labs, Palo Alto, CA, USA).

When another example specifically colored with the multimer T lymphocytes under Application of biotin / avidin-affinity interactions instead be separated from fluorescence, by further Konjugati on of the TCR-bound complex to biotin, followed by use an avidin affinity matrix. This further labeling of the multimeric complex can be indirect using a biotin-conjugated antibody that is responsible for an epitope of the fusion protein is specific. Alternatively, it can one the multimer itself either chemically or, after technical Incorporation of a BirA substrate peptide into the complex (typically the fusion protein), enzymatically conjugated to biotin in advance.

Rehearse, which in terms of antigen-specific T cells according to the methods can be enriched in the present invention, in for the study of specific interactions of antigen-specific T cells with antigen-presenting Cells, cytotoxic targets, B cells or other cellular elements of the immune system. Samples which are antigen specific T cells according to the method of the present invention may also be added used in vitro to modify such interactions; see, for. Dal., Et al., Et al., Proc. Natl. Acad. Sci. USA 90: 6671-6675 (1993).

Rehearse, which in terms of antigen-specific T cells according to the method of the present invention may also be added for therapeutic In vivo, such as for tumor immunotherapy; see, for. B. Oelke et al., Clin. Cancer Res. 6 (5): 1997-2005 (2000). Conversely, samples, which with regard to particular antigen-specific T cells according to the method depleted in the present invention, advantageously administered to patients where there is an infusion of blood fractions containing T cells, which this antigen specificity could lead to illness or morbidity.

When an alternative application or utility for the fluorescent Detection of peptide-specific T cells can be the multimeric protein complexes The present invention can also be used to advantage Peptide antigen-specific, MHC-dependent activation of T cells to stimulate.

As it is known in the art are complexes containing several with peptide loaded MHC molecules more effective than non-complexed, monomeric MHC-peptide combinations for the Stimulation of T cells that produce peptide by an MHC-dependent mechanism bind specifically; See, for example, Stryhn, A., et al., "Preformed Purified peptides / major histocompatibility class I are are potent stimulators of class I-restricted T cell hybridomas ", Eur. J. Immunol., 24 (6): 1404-9 (June 1994); Cochran, J.R., et al., "The relationship of MHC-peptide binding and T cell activation probed using chemically defined MHC class II oligomers ", Immunity, 12 (3): 241-50 (March 2000); and Piccirillo, C.A., Shevach, E.M., "Cutting edge: control of CD8 + T cell activation by CD4 + CD25 + immunoregulatory cells ", J. Immunol., 167 (3): 1137-40 (August 2001), the disclosure of which is incorporated herein by reference becomes.

tetramers MHC peptide presentation units, which is formed by interaction of the multimerization domains which are within the DsRed subunits of the multimeric complexes of present invention can therefore be loaded with peptide and incubated with a mixed population of T cells. Subpopulations of T cells carrying T cell receptors, the for specifically binding the peptide in an MHC-dependent manner are capable are activated. The greater effectiveness the tetramer compared to monomers, dimers or trimers, facilitates the activation of T cells using a lower concentration of peptide-MHC complexes. This lowers the Cost of reagents and reduces an erroneous activation, which high levels of activating peptide-MHC complexes is caused. Alternatively, it facilitates the greater effectiveness of tetrameric activation of T cell subpopulations containing receptors with lower affinity for the peptide wear.

According to one alternative embodiment may be clonal Populations of T cells with defined peptide specificity all using lower concentrations of tetrameric peptide-MHC complexes to be activated.

Thus, in another aspect, the present invention provides a method of activating peptide antigen-specific MHC-dependent T cells, which comprises contacting a T cell with a self-fluorescent self-multimeric multimeric complex of the present invention which bound the peptide during a sufficient time to effect activation.

Kits

The Compositions of the multimeric complex of the present invention can usefully be provided in the form of kits, which the execution of the Process of the present invention facilitate. Thus, the sees present invention, in another aspect, kits, which Self-fluorescent multimeric complexes of the given Invention.

In one embodiment, the kits of the present invention, as separate compositions, include a self-fluorescent (F ₁ S ₁ ) _n multimer of the present invention and an antigenic peptide. As noted above, the choice of peptide will depend on the specificity desired for the labeling reagent.

In some embodiments, a further composition comprising a second, negative control peptide is usefully included. When the negative control peptide is used to load the self-fluorescing (F ₁ S ₁ ) _n -multimer of the kit, it results in a multimeric complex that has been shown in advance to have no significant percentage of T lymphocytes in the sample to be tested labeled, thereby providing a measure of the background attributable to non-specific binding of the multimer to cells in the sample.

In other embodiments For example, the kit will include at least one pan T or T lymphocyte subgrouping antibody. As above mentioned, is meant by "pan-T antibody" an antibody, which a surface marker or recognize an epitope on all or substantially all T lymphocytes is present. By "T cell subgrouping antibody" is meant an antibody which binds to a surface marker, which is present on less than all T lymphocytes.

When the self-fluorescent multimer has been pre-loaded with peptide, such kits will typically include, as separate compositions, a multimer and at least one pan-T or sub-grouping antibody; if the self-fluorescing multimer is provided without prior peptide loading (ie, as a (F ₁ S ₁ ) _n structure), the kit will usefully become, as separate compositions, a self-fluorescent multimer, a peptide, and at least one pan-T or subgrouping antibody.

Pan-T and subgrouping antibodies which usefully included in such kits include those which are specific for CD3, CD4, CD8, CD45RO, CD45RA and CD27 are. As it will be understood the antibodies typically for the surface marker specific be as expressed by taxonomic T lymphocytes Species whose T lymphocytes be recognized by the self-fluorescent multimeric complex (e.g., anti-mouse CD3 antibody in a kit that includes a multimeric complex, the murine MHC peptide-presenting Structural units and soluble Includes derivatives).

In other embodiments the kit will contain at least one antibody to a T cell activation antigen lock in. T-cell activating antigens useful in such Kits are included, close those for CD69, CD25, CD71 and, to mark human T lymphocytes, HLA-DR specific are. Such kits can also advantageously a pan-T and / or T-cell subgrouping antibody, such as described above.

In Kits, which antibodies lock in, become the antibodies in more useful Provided in advance directly conjugated to a fluorophore, typically a fluorophore which can be differentiated by flow cytometry is from the emission of the self-fluorescent multimeric T cell-labeling complex and from the emission of other fluorophores used in the kit. Fluorophores can in more useful Way selected from the group which are fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyll-protein (PerCP), allophycocyanin (APC), Texas Red, Alexa Fluor 488 (Molecular Probes, Inc., Eugene Or.) and the tandem fluorophores PerCP-Cy5.5, PE-Cy5, PE-Cy7 and APC-Cy7 consists. antibody in the kits of the present invention can also usefully conjugated to biotin, resulting in a second-stage detection below Use of labeled strepavidin allowed.

In Kit embodiments, which include antibodies are usefully also isotype control antibody contain; As is well known, such isotype control antibodies are antibodies of identical isotype to the fluorophore-conjugated antibodies, which but directed against irrelevant specificities such as KLH, suitable for providing measurements of background binding and fluorescence.

Other kit embodiments further include a red cell lysing agent as described, inter alia, in Chang et al., U.S. Patent Nos. 4,902,613 and 4,654,312; lysie agents are well known in the art and are commercially available from a number of vendors (FACS Lysing Solution, Becton Dickinson Biosciences Immunocytometry Systems, San Jose, Calif., USA; Cal-Lysis ^TM Lysis Solution, Caltag Labs, Burlingame, CA). USA; "No-Wash" Lysis Solution, Beckman Coulter, Inc., Fullterton, CA).

Lots the kit embodiments Further instructions are provided for labeling (staining) with the multimeric complex and for execution a flow cytometric analysis.

Other Kit embodiments also close an antibody to specifically recognize and bind DsRed, or other GFP-like ones Chromophores, wherein the antibody conjugates to a fluorophore is. That way you can indirect immunofluorescence techniques. For example becomes, in a first step, the self-fluorescent, multimeric Protein complex of the present invention, which carries peptide, with T cells are incubated to effect specific binding of peptide to T cell receptors. Thereafter, in a second step, the fluorescently labeled, secondary Antibodies with the T cells are incubated so that the antibody bound to T cells GFP-like Chromophore binds. For the analysis becomes the fluorophore associated with the secondary antibody with light of the appropriate wavelength stimulated, which is typically, but not invariably, chosen that a substantial stimulation of fluorescence emission by the GFP-like Fluorophore is avoided.

A indirect immunofluorescence can also be accomplished in three steps become. As for that Two-step procedure, a second antibody is used to make the GFP-like Bind chromophore; however, the second antibody is not itself conjugated to a fluorophore. Instead, in a third Step, a fluorescently labeled third antibody added, which for the second antibody is specific and binds to it. During the analysis, that will conjugated to the third antibody Fluorophore excited by light of the appropriate wavelength. Therefore, kits of the present invention further comprises a second antibody which the GFP-like Recognizes chromophore, and a fluorescently labeled third Antibody, which the second antibody recognize, include.

As used herein, the term "antibody" includes whole antibodies, including, but not limited to, those of the classes IgG, IgM, IgA, IgE, and IgD; and antigen-specific antibody fragments, including, but not limited to, Fab, (Fab ') ₂ , phage display antibody (phAb) and single-chain variable region antibody (scFv).

The The following examples are given by way of illustration and not by way of limitation.

EXAMPLE 1

Construction of an expression vector for the Expression of an MHC-DsRed fusion protein.

Using standard molecular biology techniques, a DNA sequence encoding the extracellular domain (ECD) of murine H2-Ld MHC class I protein is removed from the H2-Ld cDNA and into the multiple cloning site of the vector pDsRed2-N1 (BD Biosciences-Clontech, Cat. # 6973-1) which had been digested with the enzymes HindIII and BamHI. This results in an in-frame fusion of the coding sequence of the ECD oriented at the amino-terminal part of the fusion with the coding sequence of the DsRed chromophore oriented at the carboxy-terminal part of the fusion. The construction of the fusion construct is shown schematically in FIG 1 shown.

The H2Ld-DsRed2 fusion construct is then ligated into the NdeI and NotI restriction enzyme sites of pBD1031S1, a BD Biosciences Pharmingen vector developed from PVL1393, cat. # 21201P. The pBD1031S1 vector is a dual expression vector containing a T7 RNA polymerase promoter for expression of the fusion construct in bacteria and a baculovirus polyhedrin promoter for expression of the fusion construct in a variety of insect cell lines, including Sf9 and Tni cells , contains. A scheme and a restriction enzyme map of the expression vector referred to as dSS-H2LdECD-DsRed-pBD1031S1 is in 2 shown.

To To complete the ligation reaction using standard techniques The ligated DNA was purified and used to grow E. coli bacteria transform, which then over Overnight on agar plates to be grown which ampicillin contained to select for transformed cells. Individual amp-resistant colonies are then in liquid culture to grow; Aliquots of bacteria are taken from which Plasmid DNA is isolated and purified.

Plasmid DNA is then double digested with the restriction enzymes NdeI and NotI to test for the presence of the Ld-DsRed insert in the vector. The digests are separated by electrophoresis in 0.7% agarose gel which is then ethidium dyed bromide or other fluorescent DNA dye, illuminated with UV light and photographed using a video still camera. Plasmids containing the expected 1.5 kb insert are stored for further analysis by sequencing (see below) to confirm the presence of the fusion construct insert. A photograph of an agarose gel showing, in seven plasmid clones, the presence of a 1.5 kb insert released by NdeI / NotI digestion is shown in FIG 3 shown.

Following the identification of insert-positive plasmid clones, clones are sequenced using standard techniques across the NdeI junction between the expression vector and the DNA sequence of the H2Ld-ECD to determine the absence of the signal peptide sequence and the presence of the correct amino terminal confirm coding sequence. A sequence lane of an automated sequencer, from the sequencing of a clone with the correct amino terminal coding sequence, is shown in U.S. Patent No. 5,396,066 4 shown.

EXAMPLE 2

Expression of the H2Ld-DsRed fusion protein in bacteria

Under Application of standard techniques become five clones of the H2Ld-DsRed fusion construct expression vector used to competent E.coli bacteria which contain an IPTG-inducible T7 RNA polymerase. To Growth on ampicillin LB agar plates overnight becomes colonies of transformed cells picked and grown overnight multiplied or expanded in LB media. The next day is the overnight culture at 37 ° C in fresh LB media containing IPTG (final concentration 1 mM), Cultivated for 2 more hours.

rehearse bacteria induced cultures are then removed and lysed in an SDS-containing buffer, after which the constituent proteins separated by denaturing SDS-polyacrylamide gel electrophoresis (PAGE) become. After electrophoresis, the gels are stained by Coomassie staining, after which the separate colored ones Proteins illuminated with visible light and with a video still camera for the be photographed visual analysis.

As it is in 5 For example, induction with IPTG for each of five H2Ld-DsRed fusion construct expression vectors used to transform E. coli results in the expression of a 60 kD protein representing the expected molecular mass of the H2Ld-DsRed fusion protein equivalent. In contrast, the induced 60 kD band is absent in negative controls which were not induced with IPTG ( 5 ).

As shown in 6 For example, the extent of IPTG-dependent induction depends on the E. coli strain transformed with an H2Ld-DsRed expression vector. The strains "NovaBlue" and "Origami" show strong IPTG induction of a 60 kD protein. Using standard techniques, Western blot analysis of the anti-DsRed antibody stained gel confirms that the 60 kD band contains DsRed protein (data not shown).

EXAMPLE 3

Expression of the H2Ld-DsRed fusion protein in insect cells

The H2Ld-DsRed expression vector is cotransfected with BaculoGold ^™ DNA (BD-Pharmingen, cat # 554739, formerly cat # 21100D) into Sf9 and Tni insect cells using standard techniques, which are discussed in U.S. Pat "BD BaculoGold ^™ Linearized Baculovirus DNA" Technical Data Sheet, incorporated herein by reference in its entirety, and available on the Pharmingen website (http://www.bdbiosciences.com/pharmingen/). The expression vector serves as a transfer vector which complements a deletion in the BaculoGold ^™ viral DNA to produce infectious virus which contains within its genome the DNA encoding the H2Ld-DsRed fusion protein. Three days after transfection, using standard techniques, medium is collected from the transfected cell cultures, the cells are removed, and the supernatant is used in the first of three rounds of amplification to obtain a high titer stock of infectious baculovirus.

Using standard techniques, the high titer stock is then used to infect Sf9 and Tni cells for the production of recombinant H2Ld-DsRed protein. Infected cells are solubilized with SDS buffer and analyzed by SDS-PAGE as described above for transformed and IPTG-induced bacteria. As shown in 7 , a 60kD protein band is expressed by infected Sf9 and Tni cells, but not expressed by uninfected negative control cells. Using standard techniques, Western blot analysis of the anti-DsRed antibody stained gel confirms that the 60kD band contains DsRed protein (data not shown).

EXAMPLE 4

Fluorescence detection of DsRed in with H2Ld-DsRed infected insect cells

Under Application of standard techniques of fluorescence microscopy Insect cells which are produced by the method described in Example 3, recombinant H2Ld-DsRed fusion construct baculovirus with regard to the expression of the H2Ld-DsRed fusion protein analyzed. From infected cells, the H2Ld-DsRed fusion protein, that contains a functional DsRed chromophore, is expected to emit red light by fluorescence emission.

8th shows uninfected negative control Sf9 cells after three days of growth in culture by conventional light microscopy. As in 9 As shown, negative control Sf9 cells do not produce red fluorescence emission when stimulated and analyzed by fluorescence microscopy three days after infection.

In contrast, the shows 10 Sf9 cells infected with recombinant H2Ld-DsRed fusion construct baculovirus by conventional light microscopy after three days growth in post-infection culture. As in 11 As shown, infected Sf9 cells emitted red fluorescent light when stimulated and analyzed by fluorescence microscopy three days after infection. Fluorescent emission of red light indicates that functional (and thus almost certainly tetramerized) DsRed chromophore is produced by the Sf9 cells infected with the H2Ld-DsRed fusion.

Similar results are observed with Tni insect cells. The 12 Figure 4 shows Tni cells infected with the recombinant H2Ld-DsRed fusion construct baculovirus by conventional light microscopy after three days growth in post-infection culture. As in 13 As shown, Tni cells infected red fluorescent light when stimulated and analyzed by fluorescence microscopy three days after infection. Fluorescent emission of red light indicates that functional DsRed chromophore is produced by the infected Tni cells.

Claims (22)

Recombinant fusion protein comprising: one GFP-like chromophore, which is a self-fluorescent Protein structural unit comprising an 11-stranded β-barrel structure with a central α-helix, where the central α-helix is a conjugated π resonance system which has two aromatic ring systems and the bridge in between includes; at least one multimerization domain, the a homomultimerization or heteromultimerization causes and for the noncovalent or covalent association of the individual subunits the fusion protein contributes; and an MHC peptide-presenting Structural unit that forms part of a protein chain of the major histocompatibility complex is missing a transmembrane domain and who participated in the presentation of peptide antigen can participate in a T cell receptor. nucleic acid comprising: a sequence encoding the fusion protein of claim 1 or to a sequence encoding the fusion protein of claim 1, complementary is. Recombinant vector comprising: the nucleic acid of Claim 2. A recombinant vector according to claim 3, wherein the vector can control the expression of the fusion protein in a host cell. A host cell, wherein the host cell comprises: One or more fusion proteins according to claim 1, the nucleic acid according to claim 2, the vector according to claim 3 and the recombinant vector according to claim 4. A self-fluorescent multimeric protein complex comprising: an amount of subunits, wherein the subunits in the complex are the quaternary formula (F) _n where F is a fusion protein according to claim 1 and n is an integer greater than 1. A self-fluorescent multimeric protein complex comprising: an amount of subunits, wherein the subunits in the complex are the quaternary formula (F ₁ S ₁ ) _n where F is a recombinant fusion protein according to claim 1; S is a soluble protein selected from the group consisting of β2 microglobulin, class II β-MHC peptide-presenting soluble derivatives, and class II α-MHC peptide-presenting soluble derivatives; S is a β2 microglobulin when F comprises a class I α-MHC peptide-presenting moiety, S is a class II β-MHC peptide-presenting soluble derivative when F is a class II α-MHC peptide-presenting moiety and S is a class II α-MHC peptide-presenting soluble derivative when F comprises a class II β-MHC peptide-presenting moiety; and n is an integer greater than 1. A self-fluorescent multimeric protein complex for labeling T lymphocytes according to the specificity of their antigen receptors, comprising: an amount of subunits, wherein the subunits in the complex are the quaternary formula (F ₁ S ₁ P ₁ ) _n where F is a recombinant fusion protein according to claim 1; S is a soluble protein selected from the group consisting of β2 microglobulin, class II β-MHC peptide-presenting soluble derivatives, and class II α-MHC peptide-presenting soluble derivatives; S is a β2 microglobulin when F comprises a class I α-MHC peptide-presenting moiety; 5 is a class II β-MHC peptide-presenting soluble derivative when F is a class II α-MHC peptide-presenting moiety and S is a class II α-MHC peptide-presenting soluble derivative when F comprises a class II β-MHC peptide-presenting moiety; P is a peptide antigen; and n is an integer greater than 1. Method for detectably labeling a T lymphocyte according to the specificity of his Antigen receptor, the method comprising: In contacting of the T lymphocyte with a self-fluorescent multimeric complex according to claim 8, wherein the antigen receptor of the T lymphocyte is specific for the peptide antigen and the MHC-presenting domains of the complex is while a time and under conditions that are sufficient to provide a detectable Allow binding of the complex to the T lymphocytes. Method for detecting T lymphocytes, which specific for a chosen one Antigens are, in a sample of cells, comprising: In contacting the sample with a self-fluorescent multimeric complex according to claim 8, wherein the peptide antigen of the complex is the chosen antigen and the MHC-presenting domains of the complex are those for which will be restricted to the T-lymphocytes that one wishes to detect, while a time and under conditions that are sufficient to provide a detectable bond the complex of T lymphocytes specific for the antigen chosen; and then Detecting specific T lymphocytes in the sample based on the fluorescence of the complex bound thereto. Method according to claim 10, further comprising: Enumerate the so proven antigen-specific T lymphocytes. Method according to claim 10, further comprising: Contacting the Sample with at least one fluorophore-conjugated antibody, wherein the antibody selected from the group is that from Pan-T antibodies and T cell subgroup antibodies consists; Detecting cell-bound fluorescence of the multimer fluorescent complex; and Detecting the cell-bound Fluorescence originating from the at least one fluorophore-conjugated antibody. Method according to claim 10, further comprising: Contacting the Sample containing at least one fluorophore-conjugated antibody, the specific to one T cell activation antigen is; and then Detecting activated specific T lymphocytes in the sample based on the fluorescence of the multimeric fluorescent complex and at least one bound thereto fluorophore-conjugated antibody. Method for enriching a sample of T-lymphocytes, specific for a chosen one Antigen, comprising: In contacting the sample with a A self-fluorescent multimeric complex according to claim 8, wherein the peptide antigen of the complex the chosen one Antigen is while a time and under conditions sufficient to provide a demonstrable bond the complex of T lymphocytes specific for the antigen chosen; and then Enrich with respect to the specific T lymphocytes based on the fluorescence of the complex bound thereto. Method for depleting a sample of T lymphocytes, specific for a chosen one Antigen, comprising: In contacting the sample with a A self-fluorescent multimeric complex according to claim 8, wherein the peptide antigen of the complex the chosen one Antigen is while a time and under conditions that are sufficient to provide a detectable bond the complex of T lymphocytes specific for the antigen chosen; and then Depleting the sample relative to the specific T lymphocytes based on the fluorescence of the complex bound thereto. Kit, the following as separate compositions includes: the multimeric complex of claim 7; and one Peptide antigen. A kit according to claim 16, which further comprises Fol gendes comprises: a fluorophore-conjugated antibody selected from the group consisting of Pan-T antibodies and T-cell subgroup antibodies. Kit according to claim 16 or 17, further comprising: a fluorophore-conjugated Antibody, specific for a T cell activation antigen is. Kit, the following as separate compositions includes: the multimeric complex of claim 8; and one fluorophore-conjugated antibody, who is selected from the group is made of Pan-T antibodies and T cell subgroup antibodies. Kit according to claim 19, further comprising: a fluorophore-conjugated Antibody, specific for a T cell activation antigen is. Kit according to one the claims 16 or 19, further comprising: a red blood cell lysing agent. A recombinant fusion protein according to claim 1, which further a flexible peptide spacer.